DNA Replication Licensing Factor Minichromosome Maintenance Deficient 5 Rescues P53-Mediated Growth Arrest

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DNA Replication Licensing Factor Minichromosome Maintenance Deficient 5 Rescues p53-Mediated Growth Arrest

Mukesh K. Agarwal, A.R.M. R. Amin, and Munna L. Agarwal

Department of Genetics, Case Western Reserve University, Cleveland, Ohio

Abstract cell cycle (22–25). In addition, p53 under certain conditions Inactivation of p53 signaling by mutation of p53 itself or protects cells by activating an additional checkpoint within S abrogation of its normal function by other transfactors, such phase (26). By using different techniques, stimuli, and cell types, as MDM2, is a key event in the development of most human laboratories around the world have identified hundreds of p53 cancers. To identify novel regulators of p53, we have used a target genes. phenotype-based selection in which a total cDNA library in a Because p53 is mutated, deleted, or inactivated in about half of all retroviral vector has been introduced into TR9-7ER cells, human cancers, we hypothesize that, in the remaining half, its which arrest when p53 is expressed from a tetracycline- ability to function as tumor suppressor may be compromised by as regulated promoter. We have isolated several clones derived yet unknown factors, negative regulators of cell growth (20). To from cells that are not growth-arrested when p53 is overex- identify novel regulators of p53, we have used a phenotype-based pressed. In one clone, the levels of p53, p21, and MDM2 are selection in which a total cDNA library in a retroviral vector has been introduced into TR9-7ER cells, which arrest when p53 is comparable with those in TR9-7ER cells and, therefore, the abrogation of growth arrest by an exogenous cDNA is likely to expressed from a tetracycline-regulated promoter (25). Clones that be distal to p21. Using reverse transcription-PCR, we were able fail to arrest may express a protein fragment from a partial cDNA to isolate a cDNA of f2.2 kb, which was found to have 99% that has a dominant-negative effect on activators of p53 activity or identity to the nucleotides between about 80 and 2,288 of the on downstream mediators of p53 function. A complete cDNA, open reading frame of a gene encoding DNA replication encoding a full-length protein that blocks p53 function when licensing factor. It encodes complete peptide of 734 residues overexpressed, should encode a negative regulator, such as the of this protein also called minichromosome maintenance known protein MDM2. We have isolated several TR9-7ER clones deficient 5 (MCM5) or cell division cycle 46 (Saccharomyces that are not growth arrested when p53 is overexpressed. The cerevisiae). Northern and Western blot analyses revealed that characterization of one such clone led to the identification of a the expression of MCM5 and its transcriptional regulator, cDNA encoding minichromosome maintenance deficient 5 (MCM5), E2F1, is negatively regulated by p53. When MCM5 cDNA was which was found to overcome p53-mediated growth arrest. reintroduced into fresh TR9-7ER cells, numerous colonies that grow in the absence of tetracycline were formed. This novel Materials and Methods observation establishes a role for MCM5 in negating the growth arrest function of p53. [Cancer Res 2007;67(1):116–21] Cell culture, plasmid transfection, and cell treatment. All cells were maintained in DMEM containing 10% fetal bovine serum and 1% penicillin

and streptomycin in a CO2 incubator. TR9-7ER cells were always Introduction maintained with 1 Ag/mL G418 and 1 Ag/mL tetracycline if otherwise not p53, a major tumor suppressor, is inactivated in most tumors indicated. TR9-7 cells were cotransfected with the plasmids containing wild- and plays a key role in mediating cellular responses to a wide type MCM5 and an empty vector containing puromycin-resistant gene variety of stimuli. In addition to external stimuli, the products of using LipofectAMINE Plus reagent according to the instruction of the several viral and cellular oncogenes also influence p53 through supplier. Individual clones were selected using puromycin. Introduction of retroviral cDNA library. Infection of TR9-7ER cells modulation of p19ARF and MDM2 (1–8). The ras/raf/mitogen- with the retroviral cDNA library was done as described (27). Briefly, high activated protein kinase signaling cascade has also been implicated titers of ecotropic virus (f107/mL) were generated following transient in regulating p53 expression (9–12). The regulation of p53 is transfection of BOSC23 packaging cells (27). After transfection with the complex, involving both the accumulation and activation of the cDNA library, supernatant medium containing the retrovirus was collected. protein in response to many distinct phosphorylation and Viral titers were estimated by infecting cells expressing the ecotropic acetylation events (13–15). It is widely believed that differently receptor with green fluorescent protein (GFP) virus generated in parallel modified forms of p53 differentially regulate patterns of gene and subjecting the cells to fluorescence-activated cell sorting analysis. Â 7 expression, which then drive distinct biological responses: cell cycle About 2 10 growing TR9-7ER cells expressing the ecotropic receptor arrest, apoptosis, control of genome integrity, regulation of DNA were infected with the supernatant medium from retrovirus-producing cells repair, etc. (16–21). An important downstream target of p53, p21/ expressing the cDNA library. Northern blot analysis. Northern blot analysis was carried out as WAF1, mediates the control by p53 of G and G checkpoints of the 1 2 described earlier (28). Total RNA was extracted with the Trizol reagent (Life Technologies, Gaithersburg, MD) as specified by the manufacturer. mRNA (0.5 Ag) or total RNA (5 Ag) was subjected to electrophoresis with a 1% Note: M.K. Agarwal and A.R.M. Ruhul Amin contributed equally to this work. agarose/formaldehyde gel and transferred to a Hybond-N nylon filter Requests for reprints: Munna L. Agarwal, Department of Genetics, Case Western (Amersham Pharmacia Biotech, Little Chalton, Buckinghamshire, England). Reserve University, Cleveland, OH 44106. Phone: 216-368-5674; Fax: 216-368-8919; The filter was hybridized with 32P-labeled cDNA probes in a rapid E-mail: [email protected]. I2007 American Association for Cancer Research. hybridization buffer (Amersham Pharmacia Biotech). Isotope intensity doi:10.1158/0008-5472.CAN-06-2835 was measured by autoradiography.

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Western blot analysis. SDS-PAGE and immunoblotting were done as spontaneous clones and that expresses ecotropic receptor for described elsewhere (29). Briefly, total cellular proteins were isolated by efficient retroviral infection. A subclone, TR9-7ER from TR9-7, was lysing the cells in 20 mmol/L Tris-HCl (pH 7.5), 2% (w/v) SDS, 2 mmol/L established after introduction of a cDNA encoding ecotropic benzamidine, and 0.2 mmol/L phenylmethylsulfonyl fluoride. Protein receptor along with puromycin resistance marker into TR9-7 cells concentrations were determined by the Bradford method (Bio-Rad, and subsequent cloning. All the cells of TR9-7ER (>2 Â 107) were Hercules, CA). Proteins were resolved on SDS-10% polyacrylamide gels and then transferred to polyvinylidene difluoride membrane. The mem- growth arrested after overexpression of p53 on withdrawal of brane was blocked in 5% nonfat skimmed milk and incubated with the tetracycline, thus indicating that the background frequency of respective antibody followed by the incubation with a secondary antibody. spontaneous clones is likely to be very low (data not shown). Proteins were visualized using enhanced chemiluminescence (Amersham Confirmation of expression of ecotropic receptor in TR9-7ER was Pharmacia Biotech) as directed by the manufacturer. accomplished by measuring green fluorescence after infection of a retroviral vector that contains cDNA encoding GFP (data not Results shown). p53 protein and its two transcriptional gene products, p21 Isolation of clones escaping p53-mediated growth arrest. and MDM2, in TR9-7ER are expressed in a time-dependent manner TR9-7 cells were originally derived from MDAH041, a fibroblast cell after tetracycline removal (Fig. 1A). line obtained from a postcrisis patient with Li-Fraumeni syndrome Gene transfer using retroviral vectors is one of the most efficient (30). There is a frameshift mutation of one of p53 allele at codon methods for introducing heritable genetic material into mamma- 184, and the normal p53 allele has been lost. No p53 protein band lian cells. We obtained a human liver cDNA library from Clontech was detected using DO-1 antibody, which recognizes both wild- (Mountain View, CA) in a retroviral vector, which was transferred type and mutant form of p53 protein (data not shown). A cDNA to TR9-7ER cells as described in Materials and Methods. p53 was encoding p53 regulated from a tetracycline-responsive promoter induced by withdrawing tetracycline 48 h after infection. Cells that was reintroduced into these cells to control the expression of p53 can grow without tetracycline are likely to express cDNAs encoding protein levels. The new cell line was termed as TR9-7, wherein a proteins that bypass the p53-mediated inhibition of cell cycle low level of wild-type p53 protein is expressed in the presence of progression. After 10 to 12 days, the growing cells were split to tetracycline (25). On withdrawal of tetracycline, the level of p53 is facilitate the expansion and growth of colonies. After 3 weeks, most induced and cells cease to grow as revealed by inhibition of the cells were growth arrested, except for nine distinct colonies. incorporation of bromodeoxyuridine into DNA (25). TR9-7 cells Photographs for growing parental cells in the presence of grown in culture might spontaneously lose p53 expression and tetracycline (a), growth-arrested parental cells after tetracycline could cause a subpopulation of cells that do not undergo p53- withdrawal (b), and that of a representative clones developed after mediated growth arrest on tetracycline withdrawal. To investigate introduction of cDNA library (c) were shown in Fig. 1B. Colonies the frequency of such loss, 2 Â 106 TR9-7 cells were grown in a that grew after tetracycline withdrawal were expanded and 25-cm plates without tetracycline. After 3 weeks, f20 colonies subjected to analysis for the expression of p53 protein and two were formed, revealing that there is a significant population of of its transcriptional targets, p21 and MDM2 (Fig. 1C). The levels of cells that have lost regulated expression of p53. To identify negative p53 and its targets are well induced after tetracycline withdrawal in regulators of p53 using cDNA library, it is essential to have a cell parental TR9-7ER cells. However, neither p53 nor p21 and MDM2 line that has either no background or extremely low background of are expressed in some clones that grow in the absence of

Figure 1. A, expression of p53, p21, and MDM2 in TR9-7ER cells. Total protein was isolated from cells grown in the absence of tetracycline (tet). Western blot analysis for p53, p21, and MDM2 using the respective antibodies. B, colonies of TR9-7ER cells that are not arrested by induced p53. a, growing TR9-7ER cells, withlow p53 in tetracycline. b, TR9-7ER cells arrested by high p53 (no tetracycline). c, colony of cells that resist arrest. C, Western blot analysis of p53, p21, and MDM2 expression in parental TR9-7ER cells and in five colonies that grow in the absence of tetracycline. A retroviral cDNA library was introduced into TR9-7ER cells. Colonies that grew after tetracycline withdrawal were expanded and subjected to analysis for the expression of p53 protein and two of its transcriptional targets, p21 and MDM2. www.aacrjournals.org 117 Cancer Res 2007; 67: (1). January 1, 2007

another clone P4-1 and parental TR9-7ER and MDAH041 cells. This increase is most likely due to the expression of an exogenous cDNA. p53-dependent down-regulation of MCM5 and E2F1. To investigate if the expression of p53 has any effect on the level of MCM5, total RNA isolated from cells after removal of tetracycline was subjected to Northern blot analysis. As shown in Fig. 3A, endogenous levels of MCM5 mRNA in p53-null MDAH041 cells were comparable with those in TR9-7ER cells, indicating that the basal level of MCM5 is not regulated by p53. However, 24 h after withdrawal of tetracycline, when the growth of cells begins to be inhibited, the level of MCM5 mRNA is highly suppressed. To further confirm the expression pattern of MCM5, cellular lysates were prepared from TR9-7ER cells after tetracycline withdrawal. Western blot analysis was done using antibodies that detect p53, p21, and MCM5. As shown in Fig. 3B, overexpression of p53 by withdrawing tetracycline led to corresponding increase in p21 and decrease in the expression of MCM5 protein. Because the promoter of MCM5 is known to have a binding site for E2F1 and p53 is known to regulate the expression of E2F1, we sought to assess the expression of E2F1. Total cellular lysates from TR9-7ER cells after tetracycline withdrawal were subjected to Western blot analysis using anti- E2F1. As shown in Fig. 3B, overexpression of p53 by tetracycline withdrawal led to down-regulation of E2F1 protein expression. MCM5 overrides p53-mediated growth arrest. To further assess if the overexpression of this exogenous cDNA was indeed responsible to confer the phenotype (i.e., to escape p53-mediated

Figure 2. Overexpression of a cDNA encoding MCM5. A, total RNA was isolated from O41 and TR9-7ER cells growing in the presence of tetracycline and clones P4-2 and P4-1 growing in the absence of tetracycline. Equal amounts of RNA were used to hybridize with 32P-tagged MCM5 (top) and glyceraldehyde- 3-phosphate dehydrogenase (GAPDH) probes (bottom). B, intensities of the band were measured by densitometry and normalized withGAPDH. tetracycline (i.e., P2-10). In some clones, p53 is expressed at a level similar to that of parental TR9-7 cells but is incapable of transactivating the expression of p21 and MDM2 (i.e., P1-1). We have two additional categories of clones: in one (P4-1), p53 seems to be degraded, as a new band of f43 kDa appears, whereas in the other (P1-3), p53 appears to be of smaller molecular weight and stabilized (accumulated). Thus, different cDNAs may be regulating the expression of functional p53 protein in different ways. Overexpression of a cDNA encoding human replication licensing factor, MCM5, in P4-2. In one clone, P4-2, the levels of p53, p21, and MDM2 are comparable with those in TR9-7ER cells. Because the expression of p53, p21, and MDM2 was not affected after the introduction of exogenous cDNA despite the abrogation of growth arrest in clone P4-2, it would likely to be distal or independent to p21 and MDM2. Using reverse transcription-PCR, we were able to isolate a cDNA of f2.2 kb. The cDNA was cloned into the sequencing/expression 3.1 vector. On sequencing, it was found to have f2,200 bp nucleotides, and on searching databases, it was found to have 99% identity to the nucleotides between about 80 and 2,288 of the open reading frame of a gene encoding DNA replication licensing factor (MCM5) protein. On a closer look, it was found to encode complete peptide of 734 residues of this protein. Figure 3. p53-dependent repression of MCM5 and E2F1. A, total RNA was To further investigate if the transcripts encoded by this isolated from O41 and TR9-7 cells growing in the presence or absence of exogenous cDNA encoding MCM5 protein are being overexpressed tetracycline (tet). The RNA was used for hybridization with probes for MCM5 in clone P4-2, expression of MCM5 mRNA was analyzed by (top) and GAPDH (bottom). Bottom of lane, fold induction of MCM5 as A normalized withGAPDH. B, tetracycline was withdrawn from TR9-7ER cells for Northern blotting. As shown in Fig. 2 , there is at least 8-fold the indicated time. Total cell lysates were used for Western blotting for p53, p21, increase in the level of MCM5 mRNA in clone P4-2 compared with MCM5, E2F1, and actin.

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Figure 4. Effect of MCM5 overexpression on p53-mediated growtharrest. A, TR9-7 cells were infected withretroviruses carrying an empty vector ( a)or containing MCM5 cDNA (b). Forty-eight hours later, tetracycline was withdrawn. Photograph was taken 12 days later. B, TR9-7 cells were transfected withplasmids containing vector (V) alone or MCM5 cDNA. Two clones (C1 and C2) were established by puromycin selection. Cells were grown in the presence or absence of tetracycline for 72 h. Total cell lysates were immunoblotted with antibodies against MCM5, p53, p21, E2F1, and actin. Bottom of lane, fold induction of MCM5. C, cells were grown in the absence of tetracycline for 12 days. The plates were stained with methylene blue. Intensities of the dye. Columns, mean of experiment done in triplicate; bars, SD. D, cells (1,000) were plated in 150-mm culture dish. Tetracycline was withdrawn after 24 h, and the numbers of colonies were counted after 3 weeks. Columns, average numbers of colonies in eachcell group done in triplicate; bars, SD. growth arrest in P4-2 cells), about 2 Â 105 growing TR9-7ER cells two clones (Fig. 4B). These results indicate that the high level were infected with the supernatant medium from retrovirus- of MCM5 in these clones is due to exogenous expression. Vector- producing cells expressing cDNA that encodes either full-length and MCM5-transfected clones were plated at a concentration of MCM5 protein or vector alone. p53 was induced by withdrawing 20,000 cells/10-cm plate. After overnight incubation, tetracycline tetracycline 48 h after infection. The plates were observed daily was withdrawn from the culture and the cells were incubated for for the next 3 weeks. The cells infected with the retroviral vector f2 weeks. The plates were then stained with methylene blue. After alone exhibited morphology similar to growth-arrested cells, staining, the dye was taken with 0.1 N HCl and dye intensity was whereas those infected with MCM5 continue to grow and became measured by spectroscopy. The experiment was done in triplicate. confluent, thus indicating that growth of MCM5-infected cells was As shown in Fig. 4C, MCM5-expressing clones showed 4- to 5-fold not inhibited despite p53 overexpression (Fig. 4A). more growth than the vector-transfected cells. Separately, vector- To unambiguously establish the role of MCM5 in overcoming and MCM5-transfected clones were plated at a concentration of p53-mediated growth arrest, we generated stable cell lines 1,000 cells/15-cm plate. After tetracycline withdrawal, cells were left expressing MCM5. TR9-7 cells were either transfected with for another 3 weeks. The plates were stained with methylene blue, pBabepuro empty vector or cotransfected with pBabepuro vector and the colonies formed in each plate were counted. The and an expression plasmid containing wild-type MCM5 in pcDNA3 experiment was done in triplicate. As shown in Fig. 4D, no colony vector. After 48 h of transfection, cells were selected with 1 Ag/mL was observed in vector-transfected cells, indicating that these cells puromycin for f2 weeks. Isolated clones were picked up and were completely growth arrested after tetracycline withdrawal. analyzed for MCM5 expression. Two independent clones expressing However, an average of 110 and 216 colonies were observed in the high level of MCM5 were isolated. To make sure that the clones are MCM5-transfected plates. still responsive to tetracycline, they were cultured in 10% DMEM in the presence of tetracycline. After overnight incubation, tetracycline was withdrawn from the culture and the cells were incubated for an Discussion additional 72 h without tetracycline. Total cell lysates were Most cancers sustain inactivation of the p53 signaling pathway. subjected to Western blotting for expression of p53, p21, E2F1, To identify novel negative regulators of p53, we made use of a and MCM5. As is shown in Fig. 4B, the levels of p53 and p21 in the phenotype-based selection approach. A complete cDNA, encoding two MCM5-expressing clones were comparable with those in a full-length protein that blocks p53 function when overexpressed, vector-transfected cells. In contrast, the expression of E2F1 should encode a negative regulator, such as the known protein decreased in both the clones and the vector-transfected cells after MDM2. We have isolated several TR9-7ER clones that are not tetracycline withdrawal. However, the level of MCM5 was >2.5-fold growth arrested when p53 is overexpressed. The levels of p53 and less after tetracycline withdrawal in vector-transfected cells, its targets are well induced after tetracycline withdrawal in whereas there was not much of a change in MCM5 level in the parental TR9-7 cells. However, neither p53 nor p21/MDM2 is www.aacrjournals.org 119 Cancer Res 2007; 67: (1). January 1, 2007

complex to origins. Each replication origin is ‘‘licensed’’ to fire once per cell cycle through the cell cycle–dependent formation and destruction of a prereplication complex (32, 33). A key component of this prereplication complex is a family of six structurally related and evolutionarily conserved proteins, MCM2-7. Biochemical studies have established the role of MCM proteins as replication licensing factors (34–36). Subsequent studies illustrate that proper orchestration of the functional interactions among MCM proteins and other components of the prereplication complex by cell cycle– dependent protein kinases results in initiation of DNA synthesis once in every cell cycle (37, 38). Despite the importance of MCMs in DNA replication and cell cycle progression, specific role of each of these MCMs is not well understood. Ryu et al. (39) observed that depletion of MCM5 in the zebrafish retina causes defects in cell cycle and apoptosis. Consistent with this study, our results suggest that ectopic expression of p53 by tetracycline withdrawal, which induces cell cycle arrest and apoptosis, is associated with striking reduction of MCM5 and overexpression of MCM5 rescues these cells from p53-induced growth inhibition. Our observations, thus, identify MCM5 as a novel transrepressional target of p53 through which p53 executes cell cycle arrest (Fig. 5). Recent studies show that the ability of p53 to transrepress certain genes is important to carry out some of its biological responses. However, the molecular Figure 5. Model: MCM5 intersects and overrides p53-mediated growtharrest function. mechanism of transrepression function of p53 remains a contro- versial area of p53 biology. Several mechanisms have been proposed, including interference with the function of transcrip- expressed in some clones that grow in the absence of tetracycline tional activators, interference with the basal transcriptional (i.e., P2-10). It is possible that the overexpression of an exogenous machinery, recruitment of chromatin-modifying factors to reduce cDNA negatively regulates the expression of p53 and its target promoter accessibility, and recruitment of transcriptional core- genes. We have two additional categories of clones: in one (P4-1), pressors (40). Moreover, transcriptional repression of certain genes p53 seems to be degraded as a new band of f43 kDa appears, by p53 can also occur indirectly through the ability of p53 to whereas in the other (P1-3), p53 appears to be of smaller molecular transactivate p21 (41–43). weight and stabilized (accumulated). In some clones, p53 is Because E2F family of transcription factors plays a pivotal role in expressed at a level similar to that of parental TR9-7 cells but is the regulation of cell cycle progression from late G1 into S phase incapable of transactivating the expression of p21 and MDM2 (i.e., (44, 45), we sought to investigate if these proteins are important in P1-1), possibly because the protein expressed from an exogenous cell cycle regulation by MCM5 and p53. The execution of the cDNA may modify p53 to render it nonfunctional in mediating the known functions of E2F1 is regulated through, at least in part, by induction of p21 and MDM2 and thus of growth arrest. Thus, controlling the expression of genes that are implicated indirectly or different cDNAs may be regulating the expression of functional p53 directly linked to cell proliferative responses (46, 47). Among these protein in different ways. It will be interesting to isolate and genes, the DNA replication licensing factors (MCM) are important. characterize the cDNAs responsible for negatively regulating the The promoter of MCM5 has multiple recognition sites for E2F1, expression or activation of the p53 protein. and the expression of MCM5 is regulated by E2F1 after serum In one clone (P4-2) where the levels of p53, p21, and MDM2 stimulation or constitutive expression of E2F1 (48). We found a remain unchanged, a cDNA encoding DNA replication licensing correlative down-regulation of MCM5 and E2F1 after expression of factor (MCM5) overcomes p53-mediated arrest. Because growth p53. p53-dependent down-regulation of MCM5 might be carried arrest function of p53 was compromised despite normal expression out via negative regulation of E2F1 (Fig. 5). of p53 and its two targets, p21 and MDM2, it is likely that MCM5 In conclusion, our genetic approach to identify negative intersects and abrogates further downstream of p21 (see model, regulators, such as MCM5, of p53 function could be used as Fig. 5). Furthermore, the expression of MCM5 is negatively diagnostic markers to elucidate the full function of p53 in regulated by p53. The reintroduction of cDNA encoding MCM5 regulation of cell cycle arrest, thus defining its tumor suppressor into fresh TR9-7 cells led to the formation of numerous colonies function more completely. Importantly, these negative regulators of that grow in the absence of tetracycline. This novel observation p53-mediated growth arrest could be used as targets for establishes a role for DNA replication licensing factor (MCM5) in therapeutic intervention. negating the growth arrest function of p53. How does MCM5 protein participate and regulate cell cycle Acknowledgments progression and cell division? In multicellular organisms, the control of genome duplication and cell division is tightly Received 8/1/2006; revised 9/15/2006; accepted 10/12/2006. Grant support: NIH grants R01 CA98916 and GM49345. coordinated. DNA replication takes place in three stages: The costs of publication of this article were defrayed in part by the payment of page prereplication complex assembly, initiation, and elongation (31). charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Prereplication complex assembly occurs during G1 and involves We thank George R. Stark and Mark W. Jackson (Cleveland Clinic Foundation, ORC-, Cdc6-, and Cdt1-dependent recruitment of the MCM2-7 Cleveland, OH) for careful reading of the article.

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Mukesh K. Agarwal, A.R.M. R. Amin and Munna L. Agarwal

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