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Clinical Cancer Research Vol. 12, 6337-6344, November 1, 2006
© 2006 American Association for Cancer Research

Human Cancer Biology

Metastasis-Suppressing Potential of Ribonucleotide Reductase Small Subunit p53R2 in Human Cancer Cells

Xiyong Liu1, Bingsen Zhou1, Lijun Xue1, Jennifer Shih1, Karen Tye1, Wesley Lin1, Christina Qi1, Peiguo Chu2, Frank Un1, Wei Wen1 and Yun Yen1

Authors' Affiliations: Departments of 1 Clinical and Molecular Pharmacology and 2 Anatomic Pathology, City of Hope National Medical Center, Duarte, California

Requests for reprints: Yun Yen, Department of Clinical and Molecular Pharmacology, City of Hope National Medical Center, 1500 East Duarte Road, Duarte, CA 91010-3000. Phone: 626-359-8111, ext. 62867; Fax: 626-301-8233; E-mail: yyen{at}coh.org.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: Previous gene transfection studies have shown that the accumulation of human ribonucleotide reductase small subunit M2 (hRRM2) enhances cellular transformation, tumorigenesis, and malignancy potential. The latest identified small subunit p53R2 has 80% homology to hRRM2. Here, we investigate the role of p53R2 in cancer invasion and metastasis.

Experimental Design: The immunohistochemistry was conducted on a tissue array including 49 primary and 59 metastatic colon adenocarcinoma samples to determine the relationship between p53R2 expression and metastasis. A Matrigel invasive chamber was used to sort the highly invasive cells and to evaluate the invasion potential of p53R2.

Results: Univariate and multivariate analyses revealed that p53R2 is negatively related to the metastasis of colon adenocarcinoma samples (odds ratio, 0.23; P < 0.05). The decrease of p53R2 is associated with cell invasion potential, which was observed in both p53 wild-type (KB) and mutant (PC-3 and Mia PaCa-2) cell lines. An increase in p53R2 expression by gene transfection significantly reduced the cellular invasion potential to 54% and 30% in KB and PC-3 cells, respectively, whereas inhibition of p53R2 by short interfering RNA resulted in a 3-fold increase in cell migration.

Conclusions: Opposite regulation of hRRM2 and p53R2 in invasion potential might play a critical role in determining the invasion and metastasis phenotype in cancer cells. The expression level of ribonucleotide reductase small subunits may serve as a biomarker to predict the malignancy potential of human cancers in the future.

Ribonucleoside diphosphate reductase (RR) plays an essential role in converting ribonucleoside diphosphate to 2'-deoxyribonucleoside diphosphate. In a RR holoenzyme, large {alpha} and small ß subunits form an {alpha}2ß2 heterotetramer that is required for RR activity (1). In humans, one large subunit (M1) and two small subunits (hRRM2 and p53R2) of RR have been identified (2). The large subunit M1 (hRRM1) contains substrate and allosteric effector sites that control the RR holoenzyme activity and substrate specificity (3–5). The RR small subunits form two equivalent dinuclear iron centers that stabilize the tyrosyl free radical required for the initiation of electron transformation during catalysis (3, 6). RR is essential as it provides deoxynucleotide triphosphate (dNTP) for DNA synthesis and DNA repair. The expression, subcellular localization, and function of RR are highly regulated (7). Because RR plays a critical role in DNA synthesis, it represents an important target for cancer therapy.

Two RR small subunits, p53R2 and hRRM2, have an 80% similarity in protein sequence (2). An in vitro assay showed that recombinant p53R2 protein, as well as hRRM2, interacts with hRRM1 to form a holoenzyme with the ability to convert CDP to dCDP (8, 9). The diiron-dityrosyl radical cluster was identified as the RR activity center, located on the common binding pockets of p53R2 and hRRM2 (10). Using a synthetic heptapeptide to inhibit RR activity, p53R2 has been shown to bind to hRRM1 through the same binding domain as hRRM2 (8). There are several different features that have been recognized in the two RR small subunits. p53R2 has been identified as a transcriptional target of the nuclear protein p53 (2, 11, 12) whereas hRRM2 was transcriptionally regulated by cell cycle–associated factors, such as NF-Y (13–15) and E2F (16). Under physiologic conditions, hRRM1 and p53R2 can be detected at the G1-G0 phase but hRRM1 does not seem to bind p53R2 or involve dNTPs synthesis in resting cells (11, 17). The nonproliferating cells do not contain RR enzymatic activity, which results in a low concentration of the dNTPs (18). In proliferating cells, the subcellular location and expression of hRRM2 is S-phase dependent (4, 18). The expression and nuclear localization of p53R2 precede that of hRRM2, which are critical for DNA synthesis in early S phase and, in turn, the physiologic growth of the cell (19). In response to DNA damage, p53R2, rather than hRRM2, was induced to facilitate DNA repair in p53 wild-type cells (2, 11, 12). However, hRRM2 can complement the p53R2 function in response to UV irradiation if p53 is dysfunctional (20). These structural and functional differences between p53R2 and hRRM2 inspired further exploration.

Malignant tumor cell growth, invasion, and subsequently metastasis may involve the alteration growth factor Ras/Raf/mitogen-activated protein kinase (MAPK) signal transduction pathway that is frequently constitutively activated by the component genes (21). The recombinant mouse RR small subunit R2 (homologous to human M2) overexpression caused an increase of membrane-associated Raf-1 expression (30%), MAPK-2 activity (70%), and Rac-1 activation (3-fold), resulting in markedly elevated metastatic potential in BALB/c 3T3 and NIH 3T3 cells (22). Those observations showed that the R2 protein was not only a rate-limiting component for ribonucleotide reduction but also capable of acting in cooperation with a variety of oncogenes, including v-fms, v-src, A-raf, v-fes, c-myc, and ornithine decarboxylase, to promote transformation and tumorigenesis (23). This has been confirmed in the human KB cancer cell line through hRRM2 overexpression (24). Of interest, the RR large subunit M1 (R1 in mice) had malignancy-suppressing potential, as shown by gene transfer experiments in both mouse and human cell lines (24, 25). p53R2 is the latest RR subunit to be identified, and its association with cell invasive potential has not yet been determined. Due to several opposing biological features between p53R2 and hRRM2, we hypothesized that p53R2 may have a malignancy-suppressing activity.

To address the above hypothesis, we investigated the expression of p53R2 among human colon cancer samples and found that p53R2 was negatively associated with the metastasis of colon adenocarcinoma. In addition, using p53R2 expression vectors and p53R2 short interfering RNA (siRNA), we showed that p53R2 could prevent cell migration in both p53-containing and p53-mutated human cancer cells.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Tissue microarray and immunohistochemical staining. The tissue microarrays of human colon adenocarcinoma (CC05-01-001) and colon metastatic adenocarcinoma (CC05-01-012) were commercially available from Cybrdi, Inc. (Frederick, MD). Details of the protocol of deparaffinization and immunohistochemistry were described on the Cybrdi website.3 Briefly, after deparaffinization, the endogenous peroxidase activity was blocked with 3% H2O2. The array slides were incubated with normal goat serum for 20 minutes, and then applied with primary antibody for 20 minutes at room temperature. After 7 minutes of hydrogen peroxide treatment, the array slides were incubated with horseradish peroxidase–labeled polymer conjugated with corresponding antibodies for 30 minutes. Then, 3,3'-diaminobenzidine (0.05 g of 3,3'-diaminobenzidine and 100 mL of 30% H2O2 in 100 mL of PBS) was applied for 5 and 10 minutes, respectively. Each slide was counterstained with hematoxylin (DAKO, Carpinteria, CA). PBS was used as a negative control. Two independent observers evaluated staining intensity to keep consistency. Five-percent positive cells were set as the positive cutoff.

A mouse polyclonal antibody against hRRM2, which was commercially produced by Convance (Princeton, NJ) using recombinant hRRM2 peptide, was used for immunohistochemical staining. The rabbit antibody against p53R2 was purchased from Alexis BioChemical company (Lausen, Switzerland) and applied for immunohistochemical staining (1:200 dilution). The mouse polyclonal antibody against p53 (Vector, Burlingame, CA) was 1:100 diluted for immunohistochemical staining. The p53 mutant stained positively whereas wild-type p53 was not detectable.

Cell culture, plasmid construction, and stable clone selection. The human oropharyngeal epidermal carcinoma KB cells (p53 wild-type), prostate PC-3 cells (p53 mutated), and pancreas Mia PaCa-2 cells (p53 mutated) were purchased from the American Type Culture Collection (Manassas, VA). KB and PC-3 cells were grown in RPMI 1640 and Mia PaCa-2 cells were grown in DMEM. The culture medium was supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin.

The construction of a sense expression vector has been described in our previous publication (24). In briefly, the cDNA of p53R2 or hRRM2 was cloned into BamH1/Not1–digested plasmid pcDNA3.1(+) (Invitrogen, San Diego, CA) to construct an expression plasmid. These plasmids were transfected into KB or PC-3 cells by electroporation. About 1 x 106 to 3 x 106 cells were trypsinized and washed with hypoosmolar electroporation buffer (Eppendorf, Westbury, NY). Cell pellets were resuspended and brought to a final volume of 400 mL in electroporation buffer containing 10 mg of the target plasmid DNA. The cell suspension was placed in an electroporation cuvette (2-mm gap, Eppendorf). Electroporation was done for 100 µs at 800 V. After 48 hours of transfection, the selection medium (300 µg/mL G-418 in RPMI 1640) was added for 4 weeks to select stabilized clones. The sense stable transfectant clones were selected and designed as KBp53R2, KBM2, PC-3p53R2, and PC-3M2.

p53R2 siRNA assay. The p53R2 siRNA (human) and scramble siRNA were commercially available from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Briefly, 2 x 105 cells were seeded per well in six-well culture plates filled with 2-mL antibiotic-free normal growth medium supplemented with fetal bovine serum, then incubated at 37°C in a CO2 incubator for 24 hours. KB or PC-3 cells were transfected with 7.2 µL of 10 µmol/L p53R2 siRNA or scramble siRNA using a transfection reagent. Cells were incubated in the transfection medium for 5 hours and then replaced with normal cell culture medium. The inhibition of p53R2 was measured by reverse transcription-PCR and Western blot.

Western blot analysis. For the Western blot analysis, the goat polyclonal antibodies against hRRM2, p53R2, and hRRM1 were obtained from Santa Cruz Biotechnology. Each 40-mg cell lysate was separated by 10% SDS-PAGE, transferred to a polyvinylidene difluoride membrane, and incubated in blocking buffer (1% I-Block reagent and 0.1% Tween 20) with the primary antibody (1:200 dilution) for 45 minutes at room temperature. After five washes, the membrane was incubated with alkaline phosphatase-secondary antibody conjugate (1:2,000 dilution) for 30 to 60 minutes. After sequential washes, each membrane was covered with a thin layer of CSPD Ready-to-Use substrate solution (Applied Biosystems, Foster City, CA), incubated for 5 minutes, and exposed to X-ray film for 3 minutes.

Cell migration and in vitro extravasation assay. Based on the modified Boyden method, we studied the invasive potential using 24-well Boyden chambers (Collaborative Research and Costar Corp., Cambridge, MA) precoated with Matrigel. In brief, the 8-mm porosity polycarbonate membrane was covered with 1 mL of medium that contained 1 x 105 cells per well. The plates were then incubated for 24 hours at 37°C in a 5% CO2 incubator. Medium was then removed and noninvading cells were gently scraped off with a cell scraper. The filter was then washed twice with PBS and then stained with 0.5% methylene blue for 4 hours. The cells that passed through the filters and adhered to the lower surface were counted by means of optical microscopy.

For in vitro extravasation assay, 1 x 105 human umbilical vein endothelial cells were seeded and incubated in EBM-2 medium (Cambrex, Walkersville, MD) for 48 hours to let the monolayer of human umbilical vein endothelial cells cover the Matrigel. Then, 1 x 105 cells per well of KB, PC-3, and their transfectants were placed into corresponding inserts and incubated with RPMI 1640 for an additional 48 hours. The cells that penetrated the human umbilical vein endothelial cell monolayer and the basement Matrigel were collected by centrifugation at 1,000 rpm and stained with 0.5% methylene blue.

Statistics. Data were collected using an MS-Excel spreadsheet. Data were analyzed using the JMP Statistical Discovery Software version 6.0 (SAS Institute, Cary, NC). Group comparisons for continuous data were done with t test for independent means or one-way ANOVA. For categorical data, we employed {chi}2 analysis, Fisher's exact test, or binomial test of proportions. Multivariate logistic regression models were used to adjust for covariate effects on the odds ratio (OR). Statistical significance was set at P < 0.05.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Expression of p53R2 is negatively related to metastasis in colon cancer samples. To investigate the relationship between p53R2 and colon cancer metastasis, immunohistochemical staining was done to detect the p53R2 protein expression level in a colon adenocarcinoma tissue microarray. The tissue microarrays were commercially available and consisted of 26 samples of nonadenocarcinoma, 59 samples of primary colon adenocarcinoma, and 49 samples of metastatic colon adenocarcinoma. The mean age of the primary was 54.9 ± 15.2 years and that of metastasis was 58.4 ± 15.0 years (t = 2.92, df = 102.9; P = 0.12). In Table 1 , it indicated that the pathologic grade was positively related to metastasis of adenocarcinoma samples (Ptrend = 0.01). There was a discrepancy in the distribution of sex between the metastasis and primary adenocarcinoma samples, which required normalization for further analysis.


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  		Table 1. Comparing expression of p53, p53R2, and hRRM2 between primary and metastatic colon adenocarcinomas

 
After immunohistochemical staining with anti-p53, anti-p53R2, and anti-hRRM2 antibodies, two pathologists examined each sample for consistency. The normal colon mucosa samples are displayed in the top row in Fig. 1 . Figure 1A shows negative staining of p53, Fig. 1B shows positive staining of p53R2, and Fig. 1C shows hRRM2 expression at crypt foci of normal mucosa, which implies that hRRM2 might dominantly contribute to cell proliferation in normal colon tissue. Of eight normal colon mucosa tissues, zero, seven, and two samples showed a positive expression of p53, p53R2, and hRRM2, respectively. The middle row in Fig. 1 contains primary colon adenocarcinoma tissue. Figure 1D shows positive expression of p53; the expression of p53 was nuclear stained as previously described. Figure 1E shows positive expression of p53R2, and Fig. 1F shows negative staining of hRRM2. The bottom row in Fig. 1 shows metastatic colon adenocarcinoma tissue. Figure 1G shows negative p53 staining, Fig. 1H shows negative staining of p53R2, and Fig. 1I shows positive expression of hRRM2, hRRM2, and p53R2, which were distinctively cytoplasmic dominant.


Figure 1
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  		Fig. 1. Immunohistochemical staining analyzes the protein level of p53, p53R2, and hRRM2 among primary and metastatic colon adenocarcinomas. Left, middle, and right, samples stained with antibody against p53, p53R2, and hRRM2, respectively. A to C, normal colon mucosa; D to F, primary colon adenocarcinoma; G to I, metastatic colon adenocarcinoma.

 
The immunohistochemistry results are summarized in Table 1. The frequencies of positive labeling of p53, p53R2, and hRRM2 were 53.3%, 81.7%, and 45.0% in primary colon adenocarcinoma samples, and 30.6%, 63.3%, and 46.9% in metastatic samples, respectively (Table 1). The ORs of metastasis for positive p53, p53R2, and hRRM2 were 0.39 [95% confidence interval (95% CI), 0.17-0.85], 0.39 (95% CI, 0.16-0.93), and 1.08 (95% CI, 0.51-2.31), respectively. These univariate results indicate that p53R2 expression is negatively related to the metastasis of colon adenocarcinoma (P < 0.05). To avoid biases resulting from confounding factors, multivariate analyses were conducted to further explore the relationship between the expression of RR small subunits and risk of colon adenocarcinoma metastasis. A multivariate unconditional logistic regression model showed that p53R2 could significantly reduce the risk of metastasis in colon adenocarcinoma. In this logistic regression model, the factors of sex and grade were taken into account to adjust the metastasis risk of p53, p53R2, and hRRM2 expression. The multivariate adjusted OR of p53R2 was 0.23 (95% CI, 0.07-0.70) and reached statistical significance in this model (P < 0.05). The ORs of p53 and hRRM2 were 0.45 (95% CI, 0.15-1.24; P = 0.129) and 1.35 (95% CI, 0.49-3.79; P = 0.559), respectively. Univariate and multivariate analysis results indicated that p53R2 reduces the risk of metastasis in colon cancer.

Interaction of p53, p53R2, and hRRM2 was further investigated. In Table 2 , the subgroup with both negatives was set as the control (OR, 1). The interaction of p53 and p53R2 might significantly reduce the OR of metastasis in colon adenocarcinomas (Ptrend < 0.05). In comparison with p53(–)/p53R2(–), the OR of p53(+)/p53R2(+) was 0.20 (95% CI, 0.06-0.62; P < 0.05). p53 could also interact with hRRM2 and increase the OR (Ptrend < 0.05); the OR of p53(+)/hRRM2(+) was 2.71 (95% CI, 0.91-8.11, P > 0.05). Because p53 could only be detected among p53-mutated samples, it was suggested that the relationship between both RR small subunits and metastasis in p53-mutated colon cancer is stronger than that in the p53 wild type. Furthermore, p53R2 was significantly related to the metastasis potential of colon adenocarcinoma (OR, 0.27; 95% CI, 0.09-0.84; P = 0.02) in hRRM2-negative groups, but not in hRRM2-positive samples (OR, 0.42; 95% CI, 0.14-1.27; P = 0.12). The above findings suggest that hRRM2 expression levels and p53 dysfunction might affect the metastasis-suppressing ability of p53R2 in colon cancer.


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  		Table 2. Correlation of combination p53, p53R2, and hRRM2 expression with metastasis potential among colon adenocarcinoma samples

 
The RR small subunit p53R2 decreases in selected highly invasive cancer cells. Because migration ability is associated with cell invasion and metastasis of cancer, the cell migration assay was employed to further evaluate the metastasis-suppressing ability of p53R2. To confirm the above findings from the human colon cancer samples, KB (oropharyngeal cancer, p53 wild-type), PC-3 (prostate cancer, p53 mutant), and Mia PaCa-2 (pancreatic cancer, p53 mutant) cell lines were employed to further investigate p53R2 and migration ability. Highly invasive cells with the ability to penetrate the Matrigel membrane were selected. Through a Western blot analysis, hRRM2 was shown to increase ~1.48-, 1.53-, and 3.93-fold in highly invasive KB, PC-3, and Mia PaCa-2 cells, respectively. p53R2 protein levels of KB, PC-3, and Mia PaCa-2 decreased to 65%, 76%, and 70%, respectively, in highly invasive cells compared with the parental cells (Fig. 2A and B ). It was indicated that hRRM2 and p53R2 might play opposite roles in regulating cell migration ability. A decrease in p53R2 and an increase in hRRM2 in highly invasive cancer cells were observed in KB, PC-3, and Mia PaCa-2 cancer cells. This result confirmed that p53R2 is negatively related to metastasis not only of colon cancers but of other cancers as well.


Figure 2
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  		Fig. 2. Western blot analysis of hRRM2 and p53R2 expression among invading cancer cells. About 2.5 x 105 cells were seeded on the Matrigel insert of a six-well invasion chamber. After incubation for 72 hours, the Matrigel inserts were removed from the chamber. The invasion cells were harvested from wells and protein was extracted for detection. A, forty micrograms of invasion cells and corresponding parental cells were separated and blotted with antibodies against hRRM2 and p53R2. The Coomassie blue stain (CBB) was employed to serve as a loading control. B, Western blot results were transferred to digital images and analyzed with ImageQuant 5.2 software.

 
Overexpression of p53R2 reduces cell migration ability in cancer cell. To investigate whether p53R2 determines invasion-suppressing ability in cancer cells, hRRM2 and p53R2 sense cDNA expression vector transfectants were employed to further evaluate the contribution of RR small subunits on cancer cell migration. Stable transfectants KBM2, KBp53R2, PC-3M2, and PC-3p53R2 were selected. Parental KB and PC-3 cells and vector transfectants served as controls. Western blot analysis results indicated that the increase in hRRM2 and p53R2 could clearly be observed in corresponding transfectants (Fig. 3A ). A cell migration assay showed that the migration ability of KBM2 and PC-3M2 increased 19.6- and 1.28-fold, respectively, in comparison with the corresponding vector controls. The cell migration ability of p53R2 transfectants was decreased to 0.54 and 0.30 in KBp53R2 and PC-3p53R2, respectively, in comparison with corresponding control vector transfectants (Fig. 3B and D). The in vitro extravasation model (Fig. 3C and D) also indicated that hRRM2 overexpression transfectants could significantly enhance the ability of cells to penetrate the human umbilical vein endothelial cell monolayer and basement membrane, whereas p53R2 overexpression transfectants could be barely seen on the bottom of wells. These studies confirmed that hRRM2 and p53R2 played opposite roles on migration ability in KB (p53 wild-type) and PC-3 (p53 mutant) cells. It also indicated that the invasion-suppressing ability of p53R2 in PC-3 cells is more significant than that in KB cells.


Figure 3
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  		Fig. 3. Migration and in vitro extravasation assays determine the invasion ability of hRRM2 and p53R2 sense expression transfectants. The sense cDNA of hRRM2 and p53R2 were inserted into a pcDNA 3.1 expression vector. The expression vectors were transfected into KB and PC-3 cells and selected with 300 and 400 µg/mL G418 for 4 weeks, respectively. A, the protein levels of hRRM2 and p53R2 among transfectants were determined by Western blot. {alpha}-Tubulin was employed to serve as a loading control. B, migration assay. About 2.5 x 104 cells were seeded on the Matrigel insert of a 24-well chamber. After incubating for 24 hours, the noninvasive cells were removed from the Matrigel inserts using a cotton-tipped swab. The invasive cells on the lower surface of the membrane were stained with 0.5% methylene blue (dissolved in 50% ethanol). C, in vitro extravasation model. About 2.5 x 104 human umbilical vein endothelial cells were grown to generate a monolayer for 48 hours on the upper side of the Matrigel. Then, KB, PC-3, and their transfectants were seeded and incubated for an additional 48 hours. The cells on the bottom were stained and counted to observe the invasive ability of the transfectants. D, the stained invading cells were counted under an optical microscope (40x) and the results are summarized.

 
Increase of cell migration ability by inhibition of p53R2. To further confirm this finding, we knocked down p53R2 expression by siRNA in KB cells. The p53R2 siRNA and scramble siRNA were available from Santa Cruz Biotechnology. After transfection for 48 hours, p53R2 expression was reduced to 40% and 50% by p53R2 siRNA, respectively, in comparison with scramble siRNA in KB and PC-3 cells (Fig. 4A ). The cells were counted and seeded into migration chambers after transfection for 24 hours, and then incubated for 48 hours for detection. The results of the cell cycle analysis indicated that p53R2 siRNA could reduce the percentage of the S phase from 12.11% to 10.79% in KB cells and from 14.34% to 12.32% in PC-3 cells (Fig. 4B). This result suggested that p53R2 siRNA did not enhance cell proliferation in both KB and PC-3 cells. Migration assay results indicated that the cell migration ability of KB and PC-3 cells was enhanced >3-fold by p53R2 siRNA (Fig. 4C). These results further confirmed that p53R2 was a component of the invasion-suppressing protein that determined the migration ability in KB and PC-3 cancer cells.


Figure 4
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  		Fig. 4. Inhibition of p53R2 by siRNA increases the invasion potential in KB and PC-3 cells. About 1 x 105 cells per well were seeded in six-well plates. KB and PC-3 cells were infected with 7.2 µL of 10 µmol/L p53R2 siRNA, or scramble siRNA, using a transfection reagent. After an incubation period of 24 hours, 1.25x104 cells per well were seeded on Matrigel chamber for migration assay. A, a Western blot showed the inhibition of p53R2 expression by siRNA after 48 and 72 hours in KB and PC-3 cells, respectively. B, flow cytometry analysis indicated that the inhibition of p53R2 slightly reduces cell proliferation in KB and PC-3 cells after being treated with p53R2 siRNA for 48 hours. C, the inhibition of p53R2 by siRNA enhances the migration ability in KB and PC-3 cells.

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
The proliferation of metastatic cancer cells requires excess dNTPs for DNA synthesis. Therefore, an increase in RR activity is necessary as it helps provide extra dNTPs for DNA replication in primary and metastatic cancer cells. In vitro, recombinant p53R2 protein, as well as hRRM2, could bind to hRRM1 to form a holoenzyme to convert CDP into dCDP (8, 9). In gene transfer experiments, the large RR subunit R1 (M1) has been found to contain a malignancy-suppressing activity (25), whereas the RR small subunit R2 (hRRM2) has been proved to play a critical role in enhancing the invasive potential in both human and mouse cells (22–24). Both hRRM1 and hRRM2 sense expression transfectants contained increased RR activities and expended dNTP pools (22, 24–26). It was concluded that the opposing roles of hRRM1 and hRRM2 in malignancy were not related to their role in dNTP synthesis (25). Univariate and multivariate analyses indicated that p53R2 expression was negatively related to colon cancer metastasis, as shown by immunohistochemical staining (OR, 0.2; 95% CI, 0.07-0.70). Further analysis revealed that the invasion-suppressing ability of p53R2 is observed not only in colon cancer but also in KB (oropharyngeal cancer), PC-3 (prostate cancer), and Mia PaCa-2 (pancreatic cancer) cells. The invasion-suppressing ability of p53R2 did not seem to be related to RR enzymatic activity. Opposing regulation by two RR small subunits revealed that the expression levels of hRRM2 and p53R2 may play a critical role in the moderation of both tumor malignancy and metastasis potential in cancer cells. These findings revealed that RR subunits may be efficient biomarkers for predicting the metastatic potential of human cancers and that the manipulation of RR small subunits may also alter their malignancy potential.

The RR subunit p53R2 is a DNA damage–inducible gene in cells containing wild-type p53 (2, 11). The p53R2 gene contains a p53 binding motif in its first intron, which explains why p53R2 could only be induced in p53 wild-type cells when placed under genotoxicity stress (2, 27, 28). p53R2 could also interact with p53 and hRRM1 at the protein level in response to UV irradiation during rapid repair phase (17). The inhibition of p53R2 through siRNA significantly increased the mutation rate in TK6 cells (29). The disruption of the p53-p53R2 DNA repair system was associated with colon tumorigenesis in ulcerative colitis (30). The above findings suggest that p53 may regulate the malignancy-suppressing ability of p53R2. Yet, the results suggest that the wild-type p53 could not enhance the malignancy-suppressing ability of p53R2. A decrease of p53R2 in invading cells was observed in both p53 wild-type (KB) and p53 mutant (PC-3 and Mia PaCa-2) cells (Fig. 1). By using the hRRM2 expression vector, the invasion-suppressing ability of p53R2 was observed in both p53 wild-type (KB) and p53 mutant (PC-3) cell lines (Fig. 2). However, this ability was more apparent in PC-3 than in KB cells. From immunohistochemistry results, p53R2 may reduce the OR of metastasis significantly only in the p53-positive (mutated) subgroup (Table 2; OR, 0.26; P < 0.05) and not in the p53-negative subgroup. The above results indicate that p53R2 is negatively related to invasive and malignancy potential in both p53 wild-type and p53 mutant cells. Furthermore, the invasion-suppressing ability of p53R2 is more apparent in cancer cells with a dysfunction of p53.

Overexpression of the mouse R2 gene is associated with the Ras/Raf/MAPK signaling pathway and cooperates with a variety of oncogenes in causing malignancy (23). Nevertheless, how RR subunits regulate the signal transduction of those oncogenes remains largely unknown. Dynamic changes in RR activity and the dNTPs pool could not explain opposing roles of hRRM2 and p53R2 in the cell migration ability of those transfectants. It was shown that the oxidative damages caused by UV, {gamma}-ray, and genotoxic chemicals were primarily governed by the accumulation of free radicals (31, 32). Hydroxyl radical–induced DNA damage can activate the K-ras 4B and C-Raf-l oncogenes, which may be one potential mechanism of how oxidants contribute to carcinogenesis (33). The free radical nitric oxide as an effective signal transducer can stimulate the enzyme guanylyl cyclase, the oncoprotein p21Ras, and protein tyrosine phosphorylation. It was concluded that nitric oxide and cyclic guanosine 3',5'-monophosphate stimulate a signaling pathway involving p21Ras-Raf-1 kinase-MAPK/extracellular signal–regulated kinase kinase-extracellular signal–regulated kinase 1/2 (34). Recent study revealed that recombinant hRRM2 protein had a prooxidant potential to oxidize carboxy-H2DCF whereas p53R2 had a peroxide removal capacity (35). In a gene transfection study, an increase in p53R2 enhanced the hydroxyl free radical removal capacity and protected cells from H2O2 attacks (35), which may be associated with migration-suppressing abilities in p53R2 transfectants. Based on the above findings, it was suggested that p53R2 played a critical role in inhibiting malignancy potential by eliminating free radicals to protect cells from oxidative damage and avoiding the activation of the Ras/Raf/MAPK signaling pathway. Nevertheless, the details of this mechanism need to be addressed in future works.

Interestingly, not only does p53R2 exhibit the same malignancy-suppressing potential as hRRM1 but its expression is also coupled with hRRM1, which is at its highest during G1-S transition (data not shown). The two-hybridization assay results revealed that the binding constant of hRRM1-p53R2 was almost 2-fold stronger than hRRM1-hRRM2 in vivo (data not shown). R1 and R2 played opposing roles in regulating the invasive potential of cancerous cells, which led to the conclusion that altering the balance of R1 and R2 expression could significantly modify the transformation, tumorigenicity, and metastatic potential (22, 25). The novel findings on metastasis-suppressing ability of p53R2 may modify the conception presented above.


   Acknowledgments
 
We thank Dr. Rebecca A. Nelson for help in setting up the JMP 6.0 software for statistical analysis and Sofia Loera for conducting the immunohistochemical staining in the co-facility laboratories at City of Hope.


   Footnotes
 
Grant support: NIH grant CA 72767.

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.

Note: X. Liu and B. Zhou are co-first authors.

3 http://www.cybrdi.com/support.php. Back

Received 4/ 3/06; revised 7/14/06; accepted 8/10/06.


   References
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 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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