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Cancer Research 67, 116-121, January 1, 2007. doi: 10.1158/0008-5472.CAN-06-2835
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Molecular Biology, Pathobiology, and Genetics

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

Requests for reprints: Munna L. Agarwal, Department of Genetics, Case Western Reserve University, Cleveland, OH 44106. Phone: 216-368-5674; Fax: 216-368-8919; E-mail: munnaagarwal{at}hotmail.com.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Inactivation of p53 signaling by mutation of p53 itself or abrogation of its normal function by other transfactors, such as MDM2, is a key event in the development of most human cancers. To identify novel regulators of p53, we have used a phenotype-based selection in which a total cDNA library in a retroviral vector has been introduced into TR9-7ER cells, which arrest when p53 is expressed from a tetracycline-regulated promoter. We have isolated several clones derived from cells that are not growth-arrested when p53 is overexpressed. In one clone, the levels of p53, p21, and MDM2 are comparable with those in TR9-7ER cells and, therefore, the abrogation of growth arrest by an exogenous cDNA is likely to be distal to p21. Using reverse transcription-PCR, we were able to isolate a cDNA of ~2.2 kb, which 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. It encodes complete peptide of 734 residues of this protein also called minichromosome maintenance deficient 5 (MCM5) or cell division cycle 46 (Saccharomyces cerevisiae). Northern and Western blot analyses revealed that the expression of MCM5 and its transcriptional regulator, E2F1, is negatively regulated by p53. When MCM5 cDNA was reintroduced into fresh TR9-7ER cells, numerous colonies that grow in the absence of tetracycline were formed. This novel observation establishes a role for MCM5 in negating the growth arrest function of p53. [Cancer Res 2007;67(1):116–21]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
p53, a major tumor suppressor, is inactivated in most tumors and plays a key role in mediating cellular responses to a wide variety of stimuli. In addition to external stimuli, the products of several viral and cellular oncogenes also influence p53 through modulation of p19ARF and MDM2 (1–8). The ras/raf/mitogen-activated protein kinase signaling cascade has also been implicated in regulating p53 expression (9–12). The regulation of p53 is complex, involving both the accumulation and activation of the protein in response to many distinct phosphorylation and acetylation events (13–15). It is widely believed that differently modified forms of p53 differentially regulate patterns of gene expression, which then drive distinct biological responses: cell cycle arrest, apoptosis, control of genome integrity, regulation of DNA repair, etc. (16–21). An important downstream target of p53, p21/WAF1, mediates the control by p53 of G1 and G2 checkpoints of the cell cycle (22–25). In addition, p53 under certain conditions protects cells by activating an additional checkpoint within S phase (26). By using different techniques, stimuli, and cell types, laboratories around the world have identified hundreds of p53 target genes.

Because p53 is mutated, deleted, or inactivated in about half of all human cancers, we hypothesize that, in the remaining half, its ability to function as tumor suppressor may be compromised by as yet unknown factors, negative regulators of cell growth (20). To identify novel regulators of p53, we have used a phenotype-based selection in which a total cDNA library in a retroviral vector has been introduced into TR9-7ER cells, which arrest when p53 is expressed from a tetracycline-regulated promoter (25). Clones that fail to arrest may express a protein fragment from a partial cDNA that has a dominant-negative effect on activators of p53 activity or on downstream mediators of p53 function. A complete cDNA, encoding a full-length protein that blocks p53 function when overexpressed, should encode a negative regulator, such as the known protein MDM2. We have isolated several TR9-7ER clones that are not growth arrested when p53 is overexpressed. The characterization of one such clone led to the identification of a cDNA encoding minichromosome maintenance deficient 5 (MCM5), which was found to overcome p53-mediated growth arrest.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
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 maintained with 1 µg/mL G418 and 1 µg/mL tetracycline if otherwise not indicated. TR9-7 cells were cotransfected with the plasmids containing wild-type MCM5 and an empty vector containing puromycin-resistant gene using LipofectAMINE Plus reagent according to the instruction of the supplier. Individual clones were selected using puromycin.

Introduction of retroviral cDNA library. Infection of TR9-7ER cells with the retroviral cDNA library was done as described (27). Briefly, high titers of ecotropic virus (~107/mL) were generated following transient transfection of BOSC23 packaging cells (27). After transfection with the cDNA library, supernatant medium containing the retrovirus was collected. Viral titers were estimated by infecting cells expressing the ecotropic receptor with green fluorescent protein (GFP) virus generated in parallel and subjecting the cells to fluorescence-activated cell sorting analysis. About 2 x 107 growing TR9-7ER cells expressing the ecotropic receptor were infected with the supernatant medium from retrovirus-producing cells expressing the cDNA library.

Northern blot analysis. Northern blot analysis was carried out as described earlier (28). Total RNA was extracted with the Trizol reagent (Life Technologies, Gaithersburg, MD) as specified by the manufacturer. mRNA (0.5 µg) or total RNA (5 µg) was subjected to electrophoresis with a 1% agarose/formaldehyde gel and transferred to a Hybond-N nylon filter (Amersham Pharmacia Biotech, Little Chalton, Buckinghamshire, England). The filter was hybridized with 32P-labeled cDNA probes in a rapid hybridization buffer (Amersham Pharmacia Biotech). Isotope intensity was measured by autoradiography.

Western blot analysis. SDS-PAGE and immunoblotting were done as described elsewhere (29). Briefly, total cellular proteins were isolated by lysing the cells in 20 mmol/L Tris-HCl (pH 7.5), 2% (w/v) SDS, 2 mmol/L benzamidine, and 0.2 mmol/L phenylmethylsulfonyl fluoride. Protein concentrations were determined by the Bradford method (Bio-Rad, Hercules, CA). Proteins were resolved on SDS-10% polyacrylamide gels and then transferred to polyvinylidene difluoride membrane. The membrane was blocked in 5% nonfat skimmed milk and incubated with the respective antibody followed by the incubation with a secondary antibody. Proteins were visualized using enhanced chemiluminescence (Amersham Pharmacia Biotech) as directed by the manufacturer.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Isolation of clones escaping p53-mediated growth arrest. TR9-7 cells were originally derived from MDAH041, a fibroblast cell line obtained from a postcrisis patient with Li-Fraumeni syndrome (30). There is a frameshift mutation of one of p53 allele at codon 184, and the normal p53 allele has been lost. No p53 protein band was detected using DO-1 antibody, which recognizes both wild-type and mutant form of p53 protein (data not shown). A cDNA encoding p53 regulated from a tetracycline-responsive promoter was reintroduced into these cells to control the expression of p53 protein levels. The new cell line was termed as TR9-7, wherein a low level of wild-type p53 protein is expressed in the presence of tetracycline (25). On withdrawal of tetracycline, the level of p53 is induced and cells cease to grow as revealed by inhibition of the incorporation of bromodeoxyuridine into DNA (25). TR9-7 cells grown in culture might spontaneously lose p53 expression and could cause a subpopulation of cells that do not undergo p53-mediated growth arrest on tetracycline withdrawal. To investigate the frequency of such loss, 2 x 106 TR9-7 cells were grown in a 25-cm plates without tetracycline. After 3 weeks, ~20 colonies were formed, revealing that there is a significant population of cells that have lost regulated expression of p53. To identify negative regulators of p53 using cDNA library, it is essential to have a cell line that has either no background or extremely low background of spontaneous clones and that expresses ecotropic receptor for efficient retroviral infection. A subclone, TR9-7ER from TR9-7, was established after introduction of a cDNA encoding ecotropic receptor along with puromycin resistance marker into TR9-7 cells and subsequent cloning. All the cells of TR9-7ER (>2 x 107) were growth arrested after overexpression of p53 on withdrawal of tetracycline, thus indicating that the background frequency of spontaneous clones is likely to be very low (data not shown). Confirmation of expression of ecotropic receptor in TR9-7ER was accomplished by measuring green fluorescence after infection of a retroviral vector that contains cDNA encoding GFP (data not shown). p53 protein and its two transcriptional gene products, p21 and MDM2, in TR9-7ER are expressed in a time-dependent manner after tetracycline removal (Fig. 1A ).


Figure 1
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  		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, with low 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.

 
Gene transfer using retroviral vectors is one of the most efficient methods for introducing heritable genetic material into mammalian cells. We obtained a human liver cDNA library from Clontech (Mountain View, CA) in a retroviral vector, which was transferred to TR9-7ER cells as described in Materials and Methods. p53 was induced by withdrawing tetracycline 48 h after infection. Cells that can grow without tetracycline are likely to express cDNAs encoding proteins that bypass the p53-mediated inhibition of cell cycle progression. After 10 to 12 days, the growing cells were split to facilitate the expansion and growth of colonies. After 3 weeks, most cells were growth arrested, except for nine distinct colonies. Photographs for growing parental cells in the presence of tetracycline (a), growth-arrested parental cells after tetracycline withdrawal (b), and that of a representative clones developed after introduction of cDNA library (c) were shown in Fig. 1B. 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 (Fig. 1C). The levels of p53 and its targets are well induced after tetracycline withdrawal in parental TR9-7ER cells. However, neither p53 nor p21 and MDM2 are expressed in some clones that grow in the absence of 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 ~43 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 ~2.2 kb. The cDNA was cloned into the sequencing/expression 3.1 vector. On sequencing, it was found to have ~2,200 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.

To further investigate if the transcripts encoded by this exogenous cDNA encoding MCM5 protein are being overexpressed in clone P4-2, expression of MCM5 mRNA was analyzed by Northern blotting. As shown in Fig. 2A , there is at least 8-fold increase in the level of MCM5 mRNA in clone P4-2 compared with another clone P4-1 and parental TR9-7ER and MDAH041 cells. This increase is most likely due to the expression of an exogenous cDNA.


Figure 2
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  		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 with GAPDH.

 
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.


Figure 3
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  		Figure 3. p53-dependent repression of MCM5 and E2F1. A, total RNA was isolated from O41 and TR9-7 cells growing in the presence or absence of tetracycline (tet). The RNA was used for hybridization with probes for MCM5 (top) and GAPDH (bottom). Bottom of lane, fold induction of MCM5 as normalized with GAPDH. B, tetracycline was withdrawn from TR9-7ER cells for the indicated time. Total cell lysates were used for Western blotting for p53, p21, MCM5, E2F1, and actin.

 
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 growth arrest in P4-2 cells), about 2 x 105 growing TR9-7ER cells were infected with the supernatant medium from retrovirus-producing cells expressing cDNA that encodes either full-length MCM5 protein or vector alone. p53 was induced by withdrawing tetracycline 48 h after infection. The plates were observed daily for the next 3 weeks. The cells infected with the retroviral vector alone exhibited morphology similar to growth-arrested cells, whereas those infected with MCM5 continue to grow and became confluent, thus indicating that growth of MCM5-infected cells was not inhibited despite p53 overexpression (Fig. 4A ).


Figure 4
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  		Figure 4. Effect of MCM5 overexpression on p53-mediated growth arrest. A, TR9-7 cells were infected with retroviruses 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 with plasmids 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 each cell group done in triplicate; bars, SD.

 
To unambiguously establish the role of MCM5 in overcoming p53-mediated growth arrest, we generated stable cell lines expressing MCM5. TR9-7 cells were either transfected with pBabepuro empty vector or cotransfected with pBabepuro vector and an expression plasmid containing wild-type MCM5 in pcDNA3 vector. After 48 h of transfection, cells were selected with 1 µg/mL puromycin for ~2 weeks. Isolated clones were picked up and analyzed for MCM5 expression. Two independent clones expressing high level of MCM5 were isolated. To make sure that the clones are 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 additional 72 h without tetracycline. Total cell lysates were subjected to Western blotting for expression of p53, p21, E2F1, and MCM5. As is shown in Fig. 4B, the levels of p53 and p21 in the two MCM5-expressing clones were comparable with those in vector-transfected cells. In contrast, the expression of E2F1 decreased in both the clones and the vector-transfected cells after tetracycline withdrawal. However, the level of MCM5 was >2.5-fold less after tetracycline withdrawal in vector-transfected cells, whereas there was not much of a change in MCM5 level in the two clones (Fig. 4B). These results indicate that the high level of MCM5 in these clones is due to exogenous expression. Vector- and MCM5-transfected clones were plated at a concentration of 20,000 cells/10-cm plate. After overnight incubation, tetracycline was withdrawn from the culture and the cells were incubated for ~2 weeks. The plates were then stained with methylene blue. After staining, the dye was taken with 0.1 N HCl and dye intensity was measured by spectroscopy. The experiment was done in triplicate. As shown in Fig. 4C, MCM5-expressing clones showed 4- to 5-fold more growth than the vector-transfected cells. Separately, vector- and MCM5-transfected clones were plated at a concentration of 1,000 cells/15-cm plate. After tetracycline withdrawal, cells were left for another 3 weeks. The plates were stained with methylene blue, and the colonies formed in each plate were counted. The experiment was done in triplicate. As shown in Fig. 4D, no colony was observed in vector-transfected cells, indicating that these cells were completely growth arrested after tetracycline withdrawal. However, an average of 110 and 216 colonies were observed in the MCM5-transfected plates.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Most cancers sustain inactivation of the p53 signaling pathway. To identify novel negative regulators of p53, we made use of a phenotype-based selection approach. A complete cDNA, encoding a full-length protein that blocks p53 function when overexpressed, should encode a negative regulator, such as the known protein MDM2. We have isolated several TR9-7ER clones that are not growth arrested when p53 is overexpressed. The levels of p53 and its targets are well induced after tetracycline withdrawal in parental TR9-7 cells. However, neither p53 nor p21/MDM2 is expressed in some clones that grow in the absence of tetracycline (i.e., P2-10). It is possible that the overexpression of an exogenous cDNA negatively regulates the expression of p53 and its target genes. We have two additional categories of clones: in one (P4-1), p53 seems to be degraded as a new band of ~43 kDa appears, whereas in the other (P1-3), p53 appears to be of smaller molecular weight and stabilized (accumulated). 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), possibly because the protein expressed from an exogenous cDNA may modify p53 to render it nonfunctional in mediating the induction of p21 and MDM2 and thus of growth arrest. Thus, different cDNAs may be regulating the expression of functional p53 protein in different ways. It will be interesting to isolate and characterize the cDNAs responsible for negatively regulating the expression or activation of the p53 protein.

In one clone (P4-2) where the levels of p53, p21, and MDM2 remain unchanged, a cDNA encoding DNA replication licensing factor (MCM5) overcomes p53-mediated arrest. Because growth arrest function of p53 was compromised despite normal expression of p53 and its two targets, p21 and MDM2, it is likely that MCM5 intersects and abrogates further downstream of p21 (see model, Fig. 5 ). Furthermore, the expression of MCM5 is negatively regulated by p53. The reintroduction of cDNA encoding MCM5 into fresh TR9-7 cells led to the formation of numerous colonies that grow in the absence of tetracycline. This novel observation establishes a role for DNA replication licensing factor (MCM5) in negating the growth arrest function of p53.


Figure 5
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  		Figure 5. Model: MCM5 intersects and overrides p53-mediated growth arrest function.

 
How does MCM5 protein participate and regulate cell cycle progression and cell division? In multicellular organisms, the control of genome duplication and cell division is tightly coordinated. DNA replication takes place in three stages: prereplication complex assembly, initiation, and elongation (31). Prereplication complex assembly occurs during G1 and involves ORC-, Cdc6-, and Cdt1-dependent recruitment of the MCM2-7 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 mechanism of transrepression function of p53 remains a controversial area of p53 biology. Several mechanisms have been proposed, including interference with the function of transcriptional activators, interference with the basal transcriptional machinery, recruitment of chromatin-modifying factors to reduce promoter accessibility, and recruitment of transcriptional corepressors (40). Moreover, transcriptional repression of certain genes by p53 can also occur indirectly through the ability of p53 to transactivate p21 (41–43).

Because E2F family of transcription factors plays a pivotal role in the regulation of cell cycle progression from late G1 into S phase (44, 45), we sought to investigate if these proteins are important in cell cycle regulation by MCM5 and p53. The execution of the known functions of E2F1 is regulated through, at least in part, by controlling the expression of genes that are implicated indirectly or directly linked to cell proliferative responses (46, 47). Among these genes, the DNA replication licensing factors (MCM) are important. The promoter of MCM5 has multiple recognition sites for E2F1, and the expression of MCM5 is regulated by E2F1 after serum stimulation or constitutive expression of E2F1 (48). We found a correlative down-regulation of MCM5 and E2F1 after expression of p53. p53-dependent down-regulation of MCM5 might be carried out via negative regulation of E2F1 (Fig. 5).

In conclusion, our genetic approach to identify negative regulators, such as MCM5, of p53 function could be used as diagnostic markers to elucidate the full function of p53 in regulation of cell cycle arrest, thus defining its tumor suppressor function more completely. Importantly, these negative regulators of p53-mediated growth arrest could be used as targets for therapeutic intervention.


   Acknowledgments
 
Grant support: NIH grants R01 CA98916 and GM49345.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank George R. Stark and Mark W. Jackson (Cleveland Clinic Foundation, Cleveland, OH) for careful reading of the article.


   Footnotes
 
Note: M.K. Agarwal and A.R.M. Ruhul Amin contributed equally to this work.

Received 8/ 1/06. Revised 9/15/06. Accepted 10/12/06.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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