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Cell Growth & Differentiation Vol. 12, 243-254, May 2001
© 2001 American Association for Cancer Research

Overexpressed Human RAD50 Exhibits Cell Death in a p21WAF1/CIP1-dependent Manner

Its Potential Utility in Local Gene Therapy of Tumor1

Boo Ahn Shin2, Kyu Youn Ahn2, Hyun Kook2, Jeong Tae Koh, In Cheol Kang, Hyun Chul Lee and Kyung Keun Kim3

Research Institute of Medical Sciences [B. A. S., K. Y. A., H. K., H. C. L., K. K. K.], and Dental Science Research Institute [J. T. K., I. C. K., K. K. K.], Chonnam National University, Kwangju 501-190, South Korea


    Abstract
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Previously, mouse RAD50, one of the mammalian DNA recombination repair genes, was reported to have limited epitopic homology to p53. Here we report the functional characteristics of overexpressed human RAD50 (hRAD50). Transient transfection of hRAD50 in several cultured cells caused cytotoxicity. We established tetracycline-regulated, stable hRAD50 expression systems in SaOS-2 cells, which retain mutated p53, and in HeLa cells. After tetracycline withdrawal, cell death and multinucleated giant cells were observed with increased hRAD50 expression, and p21WAF1/CIP1 but not p53 was increased. Transient transfection of hRAD50 in HCT116 p21-/- cells caused no cytotoxicity, but there was a significantly decreased survival rate in p21+/+ cells. These cytotoxic effects of overexpressed hRAD50 in HeLa, SaOS-2, and HCT116 p21+/+ cells were partially blocked by pretreatment of cells with N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone, a pan-caspase inhibitor. When the hRAD50 expression cDNA was injected intratumorally with liposomes, it regressed or delayed tumor development in the animal model and nitric oxide synthase expression was induced in the tumor tissues that had regressed. Our results indicate that overexpressed hRAD50 has an antiproliferation activity in vitro and in vivo in a p21-dependent manner.


    Introduction
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Cells are capable of repairing and surviving DNA damage. Gene translocations, rearrangements, amplifications, and deletions arising during the repair and misrepair of the DNA lesions, such as DSBs,4 may contribute to cell transformation and tumor development (1) . Two kinds of repair pathways are suggested to participate in the mammalian DNA DSB repair: the DNA end-joining pathway and the recombinational repair pathway (2 , 3) . The mammalian homologue of a yeast DNA recombinational repair gene (the RAD52 epistasis group: RAD50–RAD57, MRE11, and XRS2) was reported, and many studies have been done to elucidate the roles of these genes (4, 5, 6) . There is also some evidence about the roles of human RAD52 and mouse RAD54 in mammalian DNA DSB repair (7 , 8) . However, targeted disruption of mouse RAD51 induced early embryonal cell death (9) , which suggests that the recombinational process is essential for mammalian development, although there is no lethality in single-cell organisms such as yeast and Escherichia coli.

The murine homologue (mRAD50) of ScRAD50 that cross-reacted with anti-p53 monoclonal antibody mAb421 has been identified by immunoprecipitation study (5) , and the human homologue (hRAD50) was cloned by a direct cDNA selection strategy focused on the chromosomal interval spanning 5q23 to 5q31 (4) . Deletions of this region are frequently observed in hematological malignancies (10) . It has been reported that ScRAD50 and hRAD50 is a Mr 153,000 protein that exhibits a limited degree of homology to structural maintenance of chromosome proteins and synaptonemal complex proteins. All of these proteins contain regions of heptad repeats, a structural motif that gives rise to a coiled coil conformation. It is also composed of an NH2-terminal ATP-binding domain that confers the ATP-dependent double-stranded DNA binding activity (4 , 11) . ScRAD50 is part of a trimeric complex called ScRAD50/ScMRE11/ScXRS2 and is involved in the early steps of homologous recombinational DNA repair (12 , 13) , but ScRAD50 and yeast Ku70/80 also participate in an end-joining pathway (14) . Previously, we observed that 293 cells that express reduced hRAD50 by antisense cDNA transfection showed more increased sensitivity than parent cells to DNA-damaging agents, such as methylmethane sulfonate, mitomycin C, and {gamma}-irradiation, but not to UVB light (11) . It was reported that embryonic cells from the mRAD50 knockout mice showed hypersensitivity to radiation (15) . These results indicate that hRAD50 and mRAD50 are involved in the repair of mammalian DNA damage.

Previously, mRAD50 was originally identified as a Mr 180,000 cardiomyocyte phosphoprotein with limited epitopic homology to p53 (5) , and we tried to observe the effects of the overexpressed hRAD50 on cell growth. In this study, we observed that overexpressed hRAD50 in cultured cells caused p53-independent but p21-dependent cell death. Furthermore, using p21-deleted HCT116 human colon carcinoma cells, we provide convincing evidence that p21WAF1/CIP1 is required for cytotoxicity to occur by overexpressed hRAD50. Flow cytometric analysis of cell death in hRAD50-overexpressed cells showed that necrosis and apoptosis were mixed. Pretreatment with pan-caspase inhibitor Z-VAD-fmk partially blocked the p21-dependent cytotoxic effect of overexpressed hRAD50, indicating the involvement of caspases in overexpressed hRAD50-induced apoptosis.

However, a lesser degree of cell death was observed in normal fibroblasts by overexpressed hRAD50 than human cancer cells as a result of lower transfection efficiencies in the primary cells. This indicates that the hRAD50 gene delivery cassette should be administered locally into the tumor tissues to avoid the minimal toxicity to normal cells. To determine the potential in vivo impact of hRAD50 delivery on tumor development, we used experimental rat and mouse models. We injected the hRAD50 gene with liposomes into the tumor mass and found that overexpressed hRAD50 by gene delivery has antitumor activity through direct cytotoxicity and the bystander effect. These results suggest that a gene delivery cassette for overexpression of hRAD50 is one of the candidates that could be used in the local gene therapy of some tumors that are refractory to conventional p53 gene therapy.


    Results
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Overexpressed hRAD50 in Cultured Cells Induces Cytotoxicity in a p21-dependent Manner.
We observed that transient transfection of a gene cassette for hRAD50 overexpression, which is derived from the pFLAG vector, caused inhibition of cell growth compared with control vector transfection after 1.5 days in several cultured human cell lines (Table 1)Citation . It has been reported that HaCaT, HeLa, and HL-60 cells possess a mutant allele of p53, but the remaining cells express wild-type p53 (16, 17, 18) . Therefore, we established two stable human cell lines that overexpressed hRAD50, the SaOS-2 cell line that retains null p53, and the HeLa cell line, using the TC-regulated (Tet-Off) gene expression system to delineate the mechanism and characteristics of cytotoxicity by overexpressed hRAD50. Selected clones were maintained in TC-containing medium to suppress the overexpression of hRAD50. After ~2–3 days of TC withdrawal from medium, cell death and multinucleated giant cells were observed (Fig. 1, B and D)Citation . Survival of 3-day, TC-withdrawn cells were markedly decreased compared with those of cells in the presence of TC (viability: HeLa, 48.2 ± 4.1% versus 87.0± 4.5%; SaOS-2, 45.6± 3.1% versus 88.5± 4.2%). After 5 days of TC withdrawal, cell viability was still decreased compared with cells in the presence of TC (HeLa, 33.1± 3.1% versus 76.6± 2.5%; SaOS-2, 34.0± 3.0% versus 78.0± 4.2%). The oversized cells contained three or more nuclei/cell and were counted to be ~20% of the total attached cell population. Because only the nuclei of these cells (Fig. 1, A–D)Citation were immunostained with an anti-hRAD50 polyclonal antibody, whole multinucleated giant cells were visualized using tubulin and nucleus staining in the HeLa (Fig. 1F)Citation , SaOS-2 (Fig. 1, H and I)Citation , and HCT116 p21-wild (Fig. 1K)Citation cells, which were transiently transfected with the hRAD50 expression cassette. These results indicate that overexpressed hRAD50 after transfection of the hRAD50 gene delivery cassette induced endoreduplication in part of the cultured cells. We performed the gene transfection experiments on normal human conjunctival and dermal fibroblasts, rat atrial fibroblasts, and aortic smooth muscle cells for characterization of the cytotoxic effect of hRAD50 on normal cells. Compared with control cells that were transfected with vector alone, the viabilities of normal fibroblasts after 2 days of transient transfection with hRAD50 were 84.5± 2.4, 83.2± 2.7, 84.0± 4.1, and 94.8± 6.9% in the human conjunctival and dermal fibroblasts, rat aortic smooth muscle cells, and atrial fibroblasts, respectively (Table 1)Citation . These values are much higher than those of several cultured human cell lines. However, this reduced cytotoxicity by overexpressed hRAD50 in the primary fibroblasts seemed to be attributable to lower transfection efficiencies in the primary cells, because the transfection efficiencies of the reporter gene (pCMV-ß-galactosidase vector) in the primary fibroblasts were much lower than those of tumor cells (data not shown).


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Table 1 Decreased cell viability in several cultured human cell lines and normal cells after transient transfection of hRAD50

 


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Fig. 1. Transfection of hRAD50 induced endoreduplication in cultured cells. Increased multinucleated giant cells (black arrow) were observed after TC withdrawal in the HeLa (A and B) and SaOS-2 (C and D) cells. These cells (B and D) were cotransfected with a hRAD50 expression cassette and G418-resistant vector using a Tet-Off Gene Expression System. Control cells (A and C), which were transfected with the pSVneo plasmid alone, showed a mononucleated pattern. Both cells (A–D) were immunostained with an anti-hRAD50 polyclonal antibody. To visualize whole multinucleated giant cells, tubulin and nuclei were stained in the HeLa (E and F), SaOS-2 (G–I), and HCT116 p21 wild (J and K) cells, which were transiently transfected with hRAD50 expression cassette (F, H, I, and K) or control pFLAG vector (E, G, and J), and observed with confocal laser scanning microscopy (E–K). It showed that overexpressed hRAD50 after transfection of hRAD50 expression cassette induced endoreduplication or nuclear fragmentation (white arrow) in the HeLa (F), SaOS-2 (H and I), and HCT116 p21-wild (K) cells. A–D, x40; E–K, x80.

 
The Western blot analysis showed the increased full-length hRAD50 and Mr 138,000 splice variant hRAD50 expression (11) after TC withdrawal (Fig. 2A)Citation . The genomic DNA of cells that were incubated in the absence of TC showed a degradation pattern and weak DNA ladder on the agarose gel (Fig. 2B)Citation . This indicates that increased hRAD50 expression after TC withdrawal caused cell death by necrosis and in part, apoptosis. However, because the mRAD50 was originally identified as a Mr 180,000 cardiomyocyte phosphoprotein with limited epitopic homology to p53 (5) , we studied the effects of overexpressed hRAD50 on p53, p21, and p27 in HeLa and SaOS-2 cells. In 3- and 5-day-incubated cells after TC withdrawal, p21 expression was increased, but there were no changes in p53 and p27 expression (Fig. 2C)Citation . Because p21 seems to be involved in the cytotoxicity by overexpressed hRAD50, we examined the effects of overexpressed hRAD50 in HCT116 p21-deleted cells (19) . Transient transfection of hRAD50 in HCT116 p21-wild cells showed a decreased cell survival rate, endoreduplication, and increased p21 expression after 1.5 days compared with that of control vector transfection, but cell death and endoreduplication were not observed in HCT116 p21-null cells (Figs. 1KCitation and 2D)Citation . Endoreduplication was observed in the recovering HCT116 p21-wild cells that were released from overexpressed hRAD50-induced initial growth arrest, but they displayed growth retardation and delayed cell death. This finding corresponded with other reports that abnormal mitosis and endoreduplication in the recovering cells were correlated with the induced level of p21 and the duration of p21 induction (20) . This means that overexpressed hRAD50 in cultured cells caused p53-independent but p21-dependent cell death. To know whether cells expressing increased p21 levels were the same cells that were overexpressing hRAD50, p21 and hRAD50 were stained simultaneously in 3-day-incubated HeLa cells after TC withdrawal using fluorescent immunocytochemistry (Fig. 2E)Citation . hRAD50 was colocalized with p21 in the HeLa cells, and it indicates that the hRAD50- and p21-expressing cells are the same populations.



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Fig. 2. Western blot analysis of HeLa or SaOS-2 cells after TC withdrawal and an electrophoretic analysis of genomic DNA of HeLa cells. Cells were incubated in the presence (+) or absence (-) of TC (2 µg/ml) in the medium for up to 7 days. A, increased full-length and smaller variant hRAD50 expression was observed after TC withdrawal. An arrow indicates the Mr 153,000-hRAD50 protein, and the lower band is a spliced Mr 138,000 hRAD50 protein. B, genomic DNA from cells incubated in the absence of TC for 3 and 5 days showed smear pattern and weak DNA ladder, whereas there was no degradation in the genomic DNA from control cells in TC-containing medium. Lane M, 100-bp DNA ladder. C, increased p21 expression was observed after TC withdrawal. There were no changes in p53 or p27 expression after TC withdrawal. Lane C, control vector-transfected HeLa cells D, transient transfection of hRAD50 in HCT116 p21-wild cells showed decreased cell survival rate after 1.5 days compared with vector-alone transfection, but cytotoxicity was not observed in HCT116 p21 null cells. {square} and {blacksquare}, cell survival rates in vector-alone and hRAD50 cDNA transfection groups, respectively. Significance of viability difference (n = 6) between hRAD50-transfected and vector-transfected HCT116 cells was tested by Student’s t test. **, P < 0.01; NS, not significant. E, hRAD50 was colocalized with p21 in the HeLa cells. Arrowhead, expression of p21. The hRAD50 in the cell was probed polyclonal rabbit anti-hRAD50 antibody (middle panel), and the p21 was probed again with monoclonal mouse anti-p21 antibody (right panel). The nucleus was then counterstained with DAPI (left panel).

 
Analyses of Overexpressed hRAD50-induced Inhibition of Cell Proliferation.
Flow cytometric analysis of cell death, using Annexin-V-Fluos and propidium iodide staining, showed that cell necrosis and apoptosis had increased in 3-day-incubated cells after TC withdrawal (Fig. 3C)Citation compared with cells grown in the presence of TC (Fig. 3B)Citation . The decreased viability of cells grown in the presence of TC (Fig. 3B)Citation compared with those of control cells without the hRAD50 overexpression plasmid (Fig. 3A)Citation suggests that hRAD50 expression may leak out in spite of suppressed hRAD50 expression by TC. Also, flow cytometric analysis of the cell cycle revealed that S phase had decreased but G1 phases had increased more in the 2-day-incubated HeLa cells after TC withdrawal than cells in the presence of TC (data not shown). Flow cytometric analysis of cell cycle and cell death of SaOS-2 cells also showed the same pattern as HeLa cells.



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Fig. 3. Flow cytometric analysis of hRAD50-overexpressed HeLa cells. Cell death was analyzed using Annexin-V-Fluos (X axis) and propidium iodide (Y axis) staining in 3-day-incubated cells. Viable cells had decreased [lower portion: 80% (B) versus 55% (C)], but necrotic cells had increased [upper portion: 20% (B) versus 45% (C)] incubated in the TC- (C) more than the cells grown in the TC+ medium (B). The more decreased viable cells grown in the TC+ (B) than those of control cells (A) without the hRAD50 overexpression plasmid [86% (A) versus 80% (B)] means that hRAD50 expression may leak out in spite of suppression of hRAD50 expression by TC. The data show one of three representative experiments. C, control cells without plasmid transfection; TC+, cells grown in the presence of tetracycline; TC-, cells grown in the absence of TC.

 
Given the potent inhibition of cell proliferation observed in hRAD50-transfected HeLa, SaOS-2, and HCT116 p21-wild cells, we sought to determine whether this inhibition of cell proliferation might be partly caused by caspase-mediated apoptosis. Pretreatment with 100 µmol of Z-VAD-fmk/well before the transient hRAD50 transfection partially blocked the cytotoxic effect of overexpressed hRAD50 in HeLa (Fig. 4A)Citation and SaOS-2 (Fig. 4B)Citation cells compared with vehicle-pretreated cells but substantially inhibited the cytotoxic effect of overexpressed hRAD50 in HCT116 p21-wild cells (Fig. 4C)Citation . It showed the involvement of caspases in overexpressed hRAD50-mediated apoptosis and indicated that cell necrosis (Z-VAD-fmk unaffected portion) and apoptosis (Z-VAD-fmk affected portion) were mixed in the hRAD50-induced inhibition of cell proliferation.



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Fig. 4. Involvement of caspases in overexpressed hRAD50-induced inhibition of cell proliferation. Pretreatment with pan-caspase inhibitor Z-VAD-fmk (100 µmol/well) before the transient transfection of hRAD50 in HeLa (A), SaOS-2 (B), and HCT116 p21-wild cells (C) caused a significantly increased cell survival rate after 3 days compared with vehicle pretreatment group (Vehicle). A significant difference in the cell survival rate between the Vehicle and the Z-VAD group is indicated by @ (@, P < 0.05; @@, P < 0.01). {square} and {blacksquare}, cell survival rates in vector-alone and hRAD50 cDNA transfection groups, respectively. It showed that the involvement of caspases in overexpressed hRAD50-mediated apoptosis and indicated that cell necrosis and apoptosis were mixed in hRAD50-induced inhibition of cell proliferation. Transient transfection of hRAD50 in these cells represented significantly decreased cell survival rate after 3 days compared with the vector alone-transfected group (*, P < 0.05; **, P < 0.01).

 
Antitumor Activity of Overexpressed hRAD50 in the Animal Model.
Because of the results obtained in hRAD50 gene transfer experiments, we tested the ability of an injected gene cassette for overexpression of hRAD50 to cause the regression of an experimental tumor growing in the flanks of animals. In the rat tumor model, the hRAD50 gene cassette (2 µg) or control vector mixed with liposomes (3 µl, each) was injected twice (1-week interval) intratumorally, starting on day 28, when the tumors were first palpable. Complete regression of 14 (of 18) established tumors was achieved within 3 weeks after the second gene injection in the hRAD50-delivered group, and they were free of lung metastasis. Of the 18 rats, 16 survived >180 days after gene injection (Fig. 5A)Citation . However, no regression of tumors was observed in the vector-alone group, and their lungs or peritoneums showed the metastatic growth of parental cells. Only 1 of 17 rats survived >180 days after gene injection (Fig. 5A)Citation .



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Fig. 5. Survival curves of experimental tumor bearing gene-delivered rats and mice. A, newborn rats received 3 x 105 RBA cells s.c. in the right flank. After 4 weeks, palpable tumor mass was observed. Control pFLAG vector (Vector alone) or hRAD50 gene delivery cassette (hRAD50, 2 µg) mixed with FuGENE 6 was injected twice (1-week interval) directly into the tumor mass. After 1 month, regression of the tumor was observed in all rats, and 16 of 18 rats survived after 180 days in the hRAD50-delivered group. In the vector-alone group, the tumor grew quickly after 1 month, and no regression of tumors was observed. Only 1 of 17 rats survived after 180 days in the vector-alone group. B, adult mice received 1 x 105 CT-26 cells s.c. in the right flank. After 10 days, a palpable tumor mass was confirmed, and we followed the same schedule as that of the rats for a gene injection experiment. In the mice, the delivery of hRAD50 could not cause the tumor to regress compared with the rat tumor model, but it significantly prolonged the survival of mice more than the vector-alone group (73± 2.2 versus 44± 3.6 days; mean± SE).

 
In the mouse model, the hRAD50 gene cassette (4 µg) or control vector mixed with liposomes (3 µl, each) was injected twice (1-week interval) intratumorally, starting on day 10, when the tumors were first palpable. After 1 month, the tumors had grown so quickly that it looked difficult for the mice in the vector-alone group to move freely, and they lived for 44± 3.6 days after their second gene injection (Fig. 5B)Citation . Conversely, in the hRAD50 gene-injected group, the mice displayed a slower tumor growth rate than in the control group, but the tumors had not regressed, and they survived for 73± 2.2 days after the second gene injection (Fig. 5B)Citation . When CT-26 tumor cells were injected s.c. into the right flanks of mice with the hRAD50 gene cassette (2 µg), it resulted in the delay of tumor growth. In the mouse model, which showed more rapid tumor growth than the rat model, the delivery of hRAD50 could not cause the tumor growing in the flank to regress compared with those in the rat model, but it prolonged the survival of mice significantly more than the vector-injected group. It seems that effects of inhibiting tumor growth by hRAD50 gene delivery are more evident in late tumors, which grow more slowly than very early tumors that grow fast.

Immunohistochemical Analysis of Regressed Tumor Tissues.
Regression of experimental rat tumors produced by the hRAD50 gene delivery was also studied by the microscopy of histological sections of tumors excised from vector-alone and hRAD50-delivered rats. Microphotographs of hematoxylin-stained sections are shown in Fig. 6 (A–D)Citation . They show that proliferating adenocarcinoma cells were abundant in the vector-injected tumor sections (Fig. 6, A and C)Citation and also expressed PCNA, which is a marker for active cycling cells (Fig. 6E)Citation . However, in the hRAD50-delivered tumor sections, tumor margins were well encapsulated, and there were fewer blood vessels than in the control section (Fig. 6B)Citation . The immune cells, such as macrophages and lymphocytes, were highly infiltrated, and there were many apoptotic cells in the regressed tumor tissue (Fig. 6D)Citation , but PCNA was not expressed in these sections (Fig. 6F)Citation . It is well known that iNOS is derived from these infiltrating immune cells, and NO is reported to contain antitumor activity when it is produced at high concentrations (21) . To test the involvement of cytotoxic NO in the regressed hRAD50 gene-injected tumor, the immunohistochemistry of iNOS expression was performed. Interestingly, there were many IgGs in the regressed tumor sections different from those of an anti-iNOS monoclonal antibody, but IgG reacted with biotinylated horse antimouse IgG. We changed the primary antibody to a rabbit anti-iNOS polyclonal antibody. Fig. 6ICitation shows iNOS expression in the regressed tumor tissues, but there was no staining in the vector-injected sections (Fig. 6H)Citation . We also examined the immunoreactivity of p53 in both sections. Compared with the positive control of human colon cancer (Fig. 6M)Citation , p53 was not observed in either tissue (Fig. 6, K and L)Citation .



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Fig. 6. Histological analyses of tumor tissues given vector-alone or the hRAD50 expression cassette. Hematoxylin-stained sections (A and B, low-power field; C and D, high-power field) and immunohistochemical analyses of PCNA (E–G), iNOS (H–J), and p53 (K–M) expression were shown. A human colon cancer specimen was included as a positive control of the PCNA (G) and p53 (M), and a bacillus Calmette Guérin injected mouse urinary bladder was used as a positive control for the iNOS (J). Proliferating cells were abundant in the vector-injected tumor sections (A and C), but in the hRAD50-delivered tumor sections (B), tumor margins were well encapsulated, and there were fewer blood vessels than in the control section (A). The immune cells, such as macrophages and lymphocytes, were highly infiltrated, and there were many apoptotic cells in the regressed tumor tissue (D). PCNA was expressed in the proliferating cells of the vector-injected tumor sections (E, arrows), but not in the hRAD50-delivered tumor sections (F). iNOS was highly expressed in the regressed tumor sections (I) but not in the vector-injected sections (H). p53 was not observed in either section (K and L) compared with human colon cancer (M). A and B, x20; C–M, x140.

 

    Discussion
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Rapid progress has been made in understanding the molecular chains of cause and effect that determine cellular responses to DNA damage (22) . Cells should have mechanisms to sense DNA damage. The identification of a DNA-protein kinase system and the ataxia telangiectasia gene for detecting radiation-induced strand breaks represents a first step toward understanding the sensor of DNA damage. Next, the signals must be transmitted to proteins, such as p53, that determine which cellular responses are activated. The responses include cell cycle arrest, apoptosis, and DNA repair, all of which relate closely to the loss of clonogenic capacity in tumor and normal cells. In this study, we observed that overexpressed hRAD50 in cultured cells is cytotoxic. However, the mRAD50 was originally identified as an Mr 180,000 cardiomyocyte phosphoprotein with limited epitopic homology to p53 (5) , and it was hypothesized, at first, that the overexpressed hRAD50 may recruit p53, resulting in the inhibition of cell growth (11) . But overexpressed hRAD50 caused cell death in the SaOS-2, HeLa, HaCaT, and HL-60 cells that express mutant p53, as well as in the 293, Chang liver, HepG2, and SK-Hep1 cells that express wild-type p53 (16, 17, 18) . The immunoprecipitation with an anti-hRAD50 antibody and immunoblot studies using an anti-p53 antibody could not detect the p53 in the vector-alone or hRAD50-delivered cells (data not shown). These observations indicate that overexpressed hRAD50 induces a p53-independent cytotoxicity in cultured cells.

The transfer of an apoptotic or lethal gene to tumors with minimal effect on normal tissues is one of the most promising strategies for cancer gene therapy. It is well known that gene transfer of the wild-type p53 into various human cancer cells induces apoptosis and suppresses growth, both in vitro and in vivo. However, p53 is the most frequently altered gene in human cancers (23) , and viral-mediated p53 gene transfer is sometimes ineffective in causing apoptosis in certain tumor cells that retain a wild-type p53 genotype (24) . It is well known that p21WAF1/CIP1 is under the transcriptional control of p53 and is thought to be an effector for the p53-mediated suppression of cell proliferation in response to intracellular signals initiated by DNA damage (25) . Also, p21 is regulated by extracellular signals, such as TGF-ß, and serves as an effector in TGF-ß-mediated growth inhibition by p53-independent manner (17) . In comparison to p21, p27 has been shown to be induced by extracellular signals for growth arrest, such as TGF-ß and cell contact, and causes a G1 cell cycle arrest when overexpressed. p27 overexpression obstructed cell entry into S phase, but it did not cause cell death (26) . On the basis of the above reports, we hypothesize that one or both of these two inhibitor molecules mediate p53-independent cytotoxicity of overexpressed hRAD50. In this study, increased expression of p21 was observed with increased hRAD50 expression, but there were no changes in p53 and p27. Also, transient transfection of human RAD50 did not cause cell death in the p21-deleted HCT116 human colon carcinoma cells, whereas it represented cytotoxicity in control wild-type p21, p53+/+ HCT116 cells. These results indicate that overexpressed hRAD50 represents inhibition of cell growth through p53-independent p21 induction, and p21 appears to be a critical effector of overexpressed hRAD50-induced cell death in cultured cells. p21 expression has been found to signal growth arrest, independent of p53, in cells undergoing differentiation (27) . Also, induction of p21 expression has been linked to growth inhibition by phenylacetate, tumor suppressor BRCA1, IFN-{gamma}, and calcitonin, as well as {gamma}-irradiation (25 , 28, 29, 30, 31) . The reduction in cell growth with calcitonin treatment is very similar to that of overexpressed hRAD50 in p21 induction, no changes of p27 level, and significant reduction of the calcitonin-mediated growth inhibition by p21 antisense oligonucleotide treatment (30) . However, we observed that overexpressed hRAD50 caused a lesser degree of cell death in normal fibroblasts than human cancer cells attributable to lower transfection efficiencies in the primary cells. This means that the cytotoxic effect of hRAD50 is also observed in normal fibroblasts, because the cytotoxic effect of hRAD50 is p21 dependent and indicates that the hRAD50 gene delivery cassette should be administered locally into the tumor tissues to avoid the minimal toxicity to normal cells. It also suggests that hRAD50 can be used for the induction of cell death in some tumors that express a wild-type p53 genotype and are refractory to conventional p53 gene therapy. In support of this hypothesis, a recent study revealed that p21 induces permanent growth arrest with markers of replicative senescence in human tumor cells lacking functional p53 (32) .

In this work, we have observed the effect of an overexpressed hRAD50 on morphological characteristics of HeLa, SaOS-2, and HCT116 p21+/+ cells. Overexpression of hRAD50 was found to induce increased p21 expression, cell death, and endoreduplication in these cells, such as the appearance of giant cells with multiple nuclei, but not in HCT116 p21-/- cells. These giant cells were known to die as a consequence of apoptosis. These results indicate that overexpressed hRAD50 represents endoreduplication in cultured cells through p21 induction. It was reported that p21 overexpression resulted in senescence-like growth arrest in cultured human cell lines and was associated with depletion of mitosis-control proteins, leading to abnormal mitosis and endoreduplication in recovering cells (20 , 32 , 33) . Also, accumulation of 2N or 4N DNA content in cells by induction of p21 indicated that events in G1 and G2-M can be inhibited by p21, and cdc2 kinase activity was reduced upon induction of p21 (32 , 34) . These reports support the function of p21 as an inducer of replicative senescence and suggest a role for p21 in coupling DNA synthesis and mitosis. p21 has been shown to be involved in both p53-dependent and -independent control of cell proliferation, differentiation, and death (27) . p21 induction caused irreversible cell cycle arrest in both G1 and G2-M and led to morphological alterations characteristic of cells undergoing replicative senescence (20 , 32) . After release from p21-induced growth arrest, cells re-entered the cell cycle but displayed growth retardation, cell death, and decreased clonogenicity. The failure to form colonies was associated with abnormal mitosis and endoreduplication in the recovering cells and was correlated with the induced level of p21 and the duration of p21 induction (20 , 33) . In this study, we have observed initial cell death after hRAD50 cDNA transfection in a p21-dependent manner and multinucleated giant cells, which appeared later from initial cell death, and our finding corresponded with other reports (20 , 33) of irreversible cell cycle arrest and characteristic morphological alterations by p21 induction.

There is a fundamental discrepancy between our results and a recent observation that after DNA damage by anticancer chemotherapeutic agents, human cells lacking p21 can initiate and often complete entire rounds of S-phase in the absence of mitosis, leading to gross nuclear abnormalities culminating in apoptosis (35) . They suggest that novel drugs that stimulate S-M uncoupling in p21-null cells but not in p21-wild cells might prove as useful in the clinic to kill cancer cells preferentially. The basic difference is that in the former, if hRAD50, one of mammalian DNA recombination repair genes and having limited epitopic homology to p53, is overexpressed in p21-wild cells, it induced p21 expression and caused S-M uncoupling in part of the cells, but in the latter, S-M uncoupling occurred mainly in p21-null cells after DNA damage by anticancer drugs. Their interpretation is that the checkpoint (G2-M) keeping cells with damaged DNA from entering mitosis is functional in these p21-null cells, whereas the checkpoint (G1-S) that would normally prevent them from reinitiating S-phase by p21 is defective. However, our interpretation is based on the roles of p21 in control of cell proliferation, differentiation, and death (27) . Furthermore, doxorubicin treatment induced the SLP and its associated terminal growth arrest in HCT 116 p21-wild cells. This response was strongly decreased but not abolished in HCT 116 null cells of p53 or p21, and high-level overexpression of p21 was sufficient to induce SLP in HT1080 fibrosarcoma cells. But the levels of p21 expressed in doxorubicin-treated cells could account for only a fraction of doxorubicin-induced SLP (36) . Also, sustained p21 induction sensitized EJ human bladder cancer cells lacking functional p53 to apoptotic cell death induced by mitomycin C, a cross-linking DNA-damaging agent (32) . On the other hand, it was reported that DNA endoreduplication by p21 induction occurred in pRb-negative but not in pRb-positive cells and suggested that pRb is a critical determinant in blocking DNA replication and in preventing endoreduplication (37) . In our results, overexpressed hRAD50 induced cytotoxicity and endoreduplication in HeLa and SaOS-2 cells, which have inactive pRb. These reports and our results indicate that p53 and p21 act as positive regulators of senescence-like terminal proliferation arrest, but their function is neither sufficient nor absolutely required for this treatment response in tumor cells, and chemotherapeutic sensitivity might exist in the treatment response among the different tumor cells.

The process of apoptosis is a critical component of normal development and homeostasis. During apoptosis, a cascade of specific cysteine proteases, the caspases, are activated and act as the dominant regulator during cell death induction. The regulation of this activation is an important focus for cell death research (38 , 39) . It was reported that activation of caspase-3 is regulated by both p21 and ILP, and one of the molecular mechanisms of death suppression by p21 is caspase-3 inactivation (40) . However, in this study, pretreatment with pan-caspase inhibitor Z-VAD-fmk partially inhibited the p21-dependent cytotoxic effect of overexpressed hRAD50, indicating the involvement of caspases in overexpressed hRAD50-mediated apoptosis. In many cells, apoptosis involves signaling through the JNK pathway. In Jurkat T cells, Fas-induced JNK activity is dependent upon activation of the caspase cascades known to be central components of the apoptotic program. It was reported that PAK signaling is necessary for JNK activation in response to Fas receptor cross-linking (41) . Inhibition of JNK activation induced by Fas does not impair cell death, as assessed by DNA fragmentation, but in some cells, defects in the ability to activate JNK or the blockade of JNK signaling pathways can inhibit apoptosis (42) . However, expression of the catalytically active COOH terminus of PAK2, which is generated through caspase action during Fas-mediated apoptosis, induces Jurkat cell apoptosis, and PAK activity is required for activation of the JNK pathway by Fas receptor in Jurkat cells. It is suggested that PAK2 may be an important component of cell death signaling (41) . We suppose that PAK activity resulting from caspase-mediated cleavage is a necessary component of p21-dependent cytotoxic effect of overexpressed hRAD50 because of the partial blocking of overexpressed hRAD50-induced cell death by pan-caspase inhibitor Z-VAD-fmk, and that PAK2 might contribute to the induction of cell death by overexpressed hRAD50. This intriguing possibility remains to be examined.

It was reported that SAHA, a novel histone deacetylase inhibitor, induces cell cycle arrest and apoptosis in human breast cancer cells, accompanied by up-regulation of the cyclin-dependent kinase inhibitors, p21WAF1/CIP1 and p27, via a p53-independent mechanism (43) . The cytotoxic effects of SAHA on breast cancer cells were manifested by G1 and G2-M cell cycle arrest and eventual apoptosis. The pan-caspase inhibitor Z-VAD-fmk blocked SAHA-induced cell death, indicating the involvement of caspases in SAHA- mediated apoptosis. In addition, SAHA modulated cell cycle and apoptosis regulatory proteins, such as pRb and other differentiation- or growth inhibition-associated genes. The reduction in cell growth and p21 induction with SAHA treatment and the involvement of caspases in SAHA-mediated apoptosis are very similar to the present results of overexpressed hRAD50-induced cell death. These results suggest that SAHA and hRAD50 might have therapeutic potential for the local treatment of human cancers.

NO generation initiates apoptotic cell death in different experimental systems. In RAW 264.7 macrophages, NO-induced oxidative DNA damage is linked to iNOS induction (44) , and the accumulation of p53 precedes apoptotic DNA fragmentation in response to endogenously or exogenously generated NO (45) . Generally, apoptotic cell death pathways can be ascribed as p53 dependent or p53 independent (46) , and there are also p53-dependent and p53-independent signaling events in NO-induced apoptosis (47) . Although the role of NO in tumor biology is still poorly understood, NO released through the iNOS by cells of the immune system, among others, is generated for long periods and exerts a cytostatic/cytotoxic effect on tumor cells (21) . Our results show that there were many apoptotic cells and certain kinds of IgG, which reacted with secondary antimouse IgG, highly infiltrated immune cells, and iNOS expression in the regressed hRAD50-delivered tumor tissues. But p53 was not observed in these tissues. Our results and these reports suggest that the overexpressed hRAD50 represented cytotoxicity through p53-independent p21 induction, caused p53-independent NO generation, resulted in NO-induced apoptosis in the gene-delivered tissues, and inhibited the proliferation and metastasis of tumor cells to surrounding tissues, similar to the bystander effect.

Although the mechanism by which delivery of hRAD50 into tumor tissues induces iNOS expression is not clear, our results indicate that local gene therapy using hRAD50 may be beneficial for proliferation disorders. Recently, we tried to observe whether local hRAD50 gene delivery can inhibit the performed in-stent neointimal hyperplasia in a porcine coronary restenosis model. Partial regression of preestablished neointimal hyperplasia and increased expression of eNOS in the neointimal area were observed by local hRAD50 gene delivery without systemic toxicity (48) .

The ScMRE11/ScRAD50/ScXRS2 complex in yeast and the MRE11/hRAD50/NBS1 complex in human cells are nucleases with manganese-dependent 3' to 5' exonuclease and single-stranded DNA endonuclease activity and function in DNA damage detection and signaling as well as in the repair of DNA DSBs. hRAD50 is especially responsible for ATP binding by these complexes (13 , 49) . hRAD50 also interacts with BRCA1 in vivo and in vitro, and BRCA1 is suggested to be important for the cellular responses to DNA damage that are mediated by these trimeric complexes (50) . The shortened telomeres and cell senescence are caused by mutations in ScRAD50 (51) , and ScRAD50 and ScRAD51 are involved in the maintenance of telomeres in the absence of telomerase (52) . Previously, we have shown that human embryonic kidney cells, which express reduced hRAD50 by stable transfection with antisense cDNA, represented a reduced resistance to ionizing radiation and alkylating agents (11) . Recently, the null mRAD50 mutation was reported to cause embryonic stem cell lethality, abnormal embryonic development, and hypersensitivity to radiation (15) . It also indicates that the mammalian MRE11/RAD50/NBS1 complex mediates the functions that are essential for cell viability, as does RAD51 (9) .

To summarize, overexpressed hRAD50 in cultured cells caused p21-dependent cell death. Furthermore, overexpressed hRAD50 in the tumor mass of an experimental rat or mouse model by local gene delivery has antitumor activity through direct cytotoxicity and the bystander effect. These observations suggest that overexpressed hRAD50 can be used with other genes, such as p21, in the treatment of some tumors that express a wild-type p53 genotype and are refractory to conventional p53 gene therapy.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Cell Lines and Transfections.
293, HeLa, SaOS-2, and HCT116 p21-wild cells were cultured in DMEM containing 10% FCS. HCT116 p21-null cells were maintained in DMEM containing 0.4 mg/ml G418 and 0.1 mg/ml hygromycin. For stable transfection of the 293 cells, 5 x 106 cells in a 75-cm2 flask were transfected by the calcium phosphate method with 10 µg of pREP4-anti-hRAD50 plasmid. Colonies surviving in the hygromycin-containing medium were isolated. Western blot analysis was performed to choose the clone that expressed low levels of hRAD50. For stable transfection of hRAD50 into HeLa and SaOS-2 cells, 5 x 106 cells in a 75-cm2 flask were transfected by the calcium phosphate method with 10 µg of ptTA-hygro plasmid (Tet-Off Gene Expression System; Clontech, Palo Alto, CA). Colonies surviving in the hygromycin-containing medium were isolated. pTRE-hRAD50 DNA, together with pSVneo plasmid, was cotransfected in the presence of TC (2 µg/ml) into one of the ptTA-expressing HeLa or SaOS-2 stable clones. Clones expressing hRAD50 were established by selection with a medium containing G418 and TC. The hRAD50 expression started to turn on after 2-day incubation of the cells in the TC-removed culture medium.

Human normal conjunctival and skin specimens were harvested within 6 h from patients undergoing pterygium excision with conjunctival autografting, after informed consent was given. Minced tissues were attached onto the culture dish and incubated in DMEM containing 10% FCS. Rat atrial fibroblasts and aortic smooth muscle cells were maintained by the same methods.

Plasmids and Chemical.
The pTRE-hRAD50 plasmid was made by inserting hRAD50 into the pTRE vector (Clontech), which has a CMV promoter. The pFLAG-hRAD50 plasmid was made by inserting hRAD50 into the pFLAG-1 vector (Kodak, Scientific Imaging System), which has a CMV promoter. The exact coding region of hRAD50 from ATG to the stop codon was used in each case. Z-VAD-fmk (Calbiochem) was dissolved in DMSO at a concentration of 20 mM.

Cell Survival.
Twenty thousand cultured cancer cells or primary fibroblasts at 75% confluency were seeded into wells of a 12-well plate and incubated for 24 h. The cells were transiently transfected with hRAD50 or control pFLAG vector using liposomes (FuGENE 6; Boehringer Mannheim, Indianapolis, IN), and ~1–2 days after transfection, the cells were counted in eight different fields, averaged, and calculated as one case. The effects of hRAD50 were evaluated by comparing the relative cell numbers to each vector control. In some experiments, cells were pretreated with Z-VAD-fmk at 100 µmol or 0.5% DMSO (vehicle) per well before transient transfection with hRAD50, and 3 days after transfection, the cells were counted and evaluated as above.

For the controls for transient transfection efficiencies between tumor cells and normal cells, pCMVß reporter vector (Clontech) was transfected to both cells, and expressions of ß-galactosidase after 1.5–2 days of transfection were measured using a ß-galactosidase staining kit (Specialty Media; Cell & Molecular Technologies, Lavallette, NJ). Tumor cells and normal cells represented ~4–5% and <2% depending on cell types, respectively. The significance of viability difference between hRAD50-transfected and vector-transfected cells or viability difference between Z-VAD-fmk and vehicle pretreatment in hRAD50-transfected cells were tested by Student’s t test.

Immunocytochemistry and Fluorescent Immunocytochemistry.
The HeLa and SaOS-2 cells were washed with TBS/Ca solution, fixed with an acetone:methanol (1:1) solution, washed, and blocked in TBS containing 5% normal goat serum for 30 min. After washing, the cells were incubated for 1 h with the antiserum for hRAD50 diluted 1:500 in TBS with 0.3% BSA. The cells were then rinsed and incubated for 30 min with the biotinylated goat antirabbit IgG and incubated sequentially for 30 min with the ABC reagents, followed by 5-min incubation with the DAB. Finally, the cells were photographed on an inverted microscope.

HeLa, SaOS-2, or HCT116 p21-wild cells were seeded in eight-well chamber slides (LabTek) and transfected with hRAD50 using liposomes (FuGENE 6). After washing with PBS, the cells were fixed with 3.7% paraformaldehyde in PBS (pH 7.4) permeabilized with 0.2% Triton-X, washed, and blocked in PBS containing 10% normal goat serum for 10 min. After washing, hRAD50 in the cells was probed by incubating with the diluted (1:500) polyclonal rabbit anti-hRAD50 antibody (11) for 1 h at room temperature. The cells were then washed with PBS containing 0.1% Tween (PBS-T) three times and visualized with Alexa Fluor 594-conjugated goat antirabbit IgG antibody (Molecular Probes, Eugene, OR) diluted in blocking solution (1:500) for 30 min. After washing with PBS-T twice, the p21 in the cells was probed again with monoclonal mouse anti-p21 antibody (1:200; Calbiochem) and visualized with Alexa Fluor 488-conjugated goat antimouse IgG antibody (Molecular Probes). The cells were washed with PBS-T twice, equilibrated in 2 x SSC, and DNase-free RNase (Boehringer Mannheim) was added to reduce the cytoplasmic staining of the RNA. The nucleus was then counterstained with DAPI (Molecular Probes) and observed with epifluorescent microscopy (Olympus IX50).

For tubulin staining, monoclonal mouse anti-{alpha}-tubulin antibody (1:100; Zymed) and Alexa Fluor 568-conjugated goat antimouse IgG antibody were used. The nucleus was then counterstained with Sytox Green (1 µmol; Molecular Probes) and observed with confocal laser scanning microscopy (Bio-Rad).

Western Blot Analysis.
Cell lysates were prepared from 293, HeLa, and SaOS-2 cells or mouse tissues, using a lysis buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 5 mM EDTA, 1 tablet/10 ml of complete protease inhibitor mixture (Boehringer Mannheim), and 1% v/v NP40. The resolved proteins (50–100 µg) were transferred to a nitrocellulose membrane and blotted with anti-hRAD50 serum, anti-p53 or anti-p21 antibody (Santa Cruz Biotechnology, Santa Cruz, CA), or anti-p27 antibody (Transduction Laboratories, Lexington, KY) and antirabbit or antimouse Ig-HRP (Ammersham, Arlington Heights, IL) as described previously (11) .

Flow Cytometric Analysis.
Single-cell suspensions from the HeLa or SaOS-2 cells were prepared and stained with propidium iodide, and the status of cell cycle parameters was measured with a FACScan flow cytometer (Becton Dickinson, Sunnyvale, CA) using a Cycle Test PLUS DNA Reagent kit (Becton Dickinson). The analysis of cell death was performed with a FACScan flow cytometer, after propidium iodide and Annexin-V-Fluos (Boehringer Mannheim) staining.

Generation of Experimental Animal Tumor Models and Effects of hRAD50 DNA Injection on Tumor Growth.
The rat tumor model was made by injecting 3 x 105 (50 µl) RBA cells s.c. into the right flanks of newborn (~1–2 days) Sprague-Dawley rats. RBA cells were derived from rat mammary adenocarcinoma (American Type Culture Collection; CRL-1747). After 4 weeks, a palpable tumor mass (diameter, <1 cm) was confirmed, and a gene expression cassette mixed with FuGENE 6 was injected directly into the tumor mass. For the mouse tumor model, 1 x 105 (50 µl) CT-26 cells were injected s.c. into the right flank of an adult BALB/c mouse (30 g). CT-26 cells were derived from mouse colon adenocarcinoma. After 10 days, a palpable tumor mass was confirmed and used for the gene injection experiment. Rats or mice were injected weekly, for 2 weeks, with a control pFLAG vector or pFLAG-hRAD50 DNA, and survival of the animals was monitored for ~3–6 months after gene injection.

Tissue Preparation and Immunohistochemistry.
Rats or mice were anesthetized with an intraperitoneal injection of sodium pentobarbital (50 mg/kg). The tumor mass was excised and immersed in 4% paraformaldehyde fixative overnight at 4°C. The tissue blocks were washed, dehydrated, embedded in paraffin, cut at 6 µm, and mounted. Immunohistochemistry was performed using an immunoperoxidase procedure (VECTA ABC kit; Vector Laboratory). The tissue sections were deparaffinized, rehydrated, rinsed, and then treated with 3% H2O2 in 60% methanol for 30 min to quench endogenous peroxidase activity. After washing, the sections were blocked in PBS containing 5% normal goat or horse serum for 1 h. The sections were incubated for 12–14 h with the antibodies for the iNOS (polyclonal; Transduction Laboratory), PCNA, and p53 (monoclonal; Santa Cruz Biotechnology) diluted in PBS with 0.3% BSA. For a negative control, the sections were incubated in PBS containing only 5% normal goat or horse serum. The sections were then rinsed and incubated sequentially for 30 min each with the biotinylated secondary antibody and the ABC reagents, followed by 5-min incubation with DAB, and were photographed on a light microscope.


    Acknowledgments
 
We thank Dr. Loren J. Field (Krannert Institute, Indiana University, Indianapolis, IN) for providing the mouse RAD50 cDNA clone, Prof. D. Y. Shin (Dankook University, Cheonan, South Korea) for providing the HCT116 cell lines, and Drs. Z. H. Lee and H-H. Kim (Chosun University, Kwangju, South Korea) for comments on the manuscript.


    Footnotes
 
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.

1 This work was supported by Korea Research Foundation Grant KRF-99-015-DP0304. Back

2 These authors contributed equally to this work. Back

3 To whom requests for reprints should be addressed, at the Department of Pharmacology, College of Dentistry, Chonnam National University, Hak-Dong 5, Dong-Ku, Kwangju 501-190, South Korea. Phone: 82-62-220-4235; Fax: 82-62-232-6974; E-mail: kimkk{at}chonnam.chonnam.ac.kr Back

4 The abbreviations used are: DSB, double-strand break; ScRAD50, yeast RAD50; hRAD50, human RAD50; mRAD50, murine RAD50; PCNA, proliferating cell nuclear antigen; iNOS, inducible nitric oxide synthase; TGF-ß, transforming growth factor-ß; TC, tetracycline; DAB, diaminobenzidine; SLP, senescence-like phenotype; JNK, c-Jun NH2-terminal kinase; PAK, p21-activated kinase; SAHA, suberoylanilide hydroxamic acid; Z-VAD-fmk, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone; CMV, cytomegalovirus. Back

Received for publication 8/22/00. Revision received 2/19/01. Accepted for publication 3/16/01.


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 Introduction
 Results
 Discussion
 Materials and Methods
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