Departments of 1Environmental Medicine, 2Pediatrics, 3Pathology and Laboratory Medicine, 4Biochemistry and Biophysics, and 5Radiation Oncology, School of Medicine and Dentistry, The University of Rochester, Rochester, New York 14642
Submitted 22 July 2003 ; accepted in final form 18 August 2003
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ABSTRACT |
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cell cycle; DNA damage; proliferation; reactive oxygen species; proliferating cell nuclear antigen; cyclin-dependent kinase
Despite these observations, the mechanisms by which hyperoxia activates p21 and subsequent inhibition of proliferation by p21 remain unclear. For example, the role of p53 in the induction of p21 during hyperoxia has not been established. It is unlikely that hyperoxia increased p21 in SV40-T2 cells through a p53-dependent process because large T antigen used to immortalize the cells blocks p53 transcriptional activity (1). Because neutralizing antibodies against transforming growth factor (TGF)- partially restored cyclin E-dependent kinase activity, it was concluded that hyperoxia induced p21 in SV40-T2 cells through TGF-
signaling (8). Although TGF-
can increase p21 transcription (9), a direct link during hyperoxia has yet to be established. Another study using HCT116 colon carcinoma cells and isogenic lines in which p53 or p21 were deleted by homologous recombination argued for p53-dependent induction of p21 during hyperoxia (15). Although efforts were made to use cells with comparable passage numbers, cell lines lacking p53 or p21 can undergo additional genetic changes over time that may modify their response to oxidative stress. In vivo animal studies have also not resolved this issue since p53-dependent and independent induction of p21 has been reported in adult mice exposed to hyperoxia (21, 23). It also remains unclear whether one or both inhibitory domains on p21 exert G1 arrest during hyperoxia. Unlike the SV40-T2 cells, hyperoxia inhibited cyclin E-dependent kinase activity in T47D-H3 cells independently of p21 (3). Moreover, the role of the carboxy-terminal PCNA binding domain in oxidative growth arrest has not been studied.
To address these issues, we assessed the effect of hyperoxia on growth of the p53-deficient human lung adenocarcinoma cell line. This cell line was chosen because it contains a large deletion in the p53 gene and does not induce p21 during genotoxic stress (37). Overexpressing p53, p21, individual domains of p21, or the related kinase inhibitor p27 allowed us to assess the contribution of each protein to exert G1 arrest during hyperoxia.
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MATERIALS AND METHODS |
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Generation of cell lines. Approximately 2 x 105 cells were seeded in a 60-mm dish and incubated overnight. PC53-SN3 plasmid (5 µg) containing a wild-type p53 cDNA fused with cytomegalovirus (CMV) promoter (2) was transfected into cells with Superfectin (Qiagen, Valencia, CA) according to the manufacturer's protocol. The medium was replaced 48 h later with DMEM containing 0.6 mg/ml of G418 (Invitrogen, Carlsbad, CA). We picked and analyzed the G418-resistant clones for the expression of p53 by Western blot analysis using anti-p53 antibody (Calbiochem, San Diego, CA). The selected clone was expanded and cultured in DMEM containing 300 µg/ml of G418.
All PCR products were amplified using a commercially available kit (Applied Biosystems, Foster City, CA). The human p21 open reading frame (ORF) was generated by RT-PCR of RNA isolated from the human lung adenocarcinoma A549 cells using forward 5'-atgtcagaaccggctg-3' and reverse 5'-ttagggcttcctcttggag-3' primers. A BamHI restriction enzyme site and Kozak consensus sequence (5'-acc-3') for translation initiation were added to the forward primer, and a HindIII restriction site was added after the termination codon of the reverse primer. The PCR product was ligated into the pCR2.1 vector with the TA cloning kit (Invitrogen). We then inserted the p21 cDNA into the pBIG2i vector (35) using BamHI and SpeI sites. We made an in-frame fusion of the p21 and the enhanced green fluorescence protein (EGFP) sequences by removing the EGFP sequence from the pEGFP-C1 vector (Clontech, Palo Alto, CA) using NheI and BamHI and inserting it into the pBIG2i p21 vector to produce the pBIG2i EGFp21 vector.
The human p27 ORF was generated by PCR of a plasmid containing the human p27 sequence (pCMV5 p27) using the forward primer 5'-atgtcaaacgtgcgag-3' and the reverse primer 5'-ttacgtttgacgtcttctg-3'. A BamHI site and a Kozak consensus sequence for translation initiation were added to the 5' end of the ORF using the forward primer, and a SpeI site was added to the 3' end using the reverse primer. We made an in-frame fusion of the EGFP and p27 sequences by liberating p21 from the pBIG2i EGFP fused amino-terminal to p21 (EGFp21) plasmid using BamHI and SpeI and ligating the p27 sequence into the resulting vector to produce pBIG2i EGFp27.
The p21 cyclin/Cdk binding domain sequence (representing amino acids 1-82) was generated by RT-PCR of RNA isolated from human colon carcinoma HCT116 cells using the forward 5'-atgtcagaaccggct-ggg-3' and reverse 5'-gggccccgtgggaaggtagag-3' primers. An EcoRI site was added to the 5' end of the sequence using the forward primer, and a nuclear localization signal (NLS) (16), stop codon, and BamHI site were added to the 3' end using the reverse primer. The EcoRI- and BamHI-digested fragment was ligated into a pBIG2i vector that contained an amino-terminal Met-Flag coding sequence upstream of the EcoRI site and the internal ribosome entry site (IRES)-EGFP sequence from pIRES2-EGFP (Clontech) downstream of the BamHI site to produce pBIG2i p21(Amino) IRES-EGFP. The p21 PCNA binding domain sequence (representing amino acids 76-164) was generated by PCR of the pBIG2i p21 plasmid described above using the forward 5'-ctctaccttcccacggggc-3' and reverse 5'-ttagggcttcctctt-ggag-3' primers. EcoRI and BamHI sites were added to the 5' and 3' ends of the sequence using their respective primers. The EcoRI- and BamHI-digested fragment was ligated into a pBIG2i vector that contained an amino-terminal Met-Flag coding sequence upstream of the EcoRI site and the IRES-EGFP sequence from pIRES2-EGFP (Clontech) downstream of the BamHI site pBIG2i p21(Carboxy) IRES-EGFP. The p21 and p27 transgenes were sequenced by ABI PRISM BigDye Terminator Cycle Sequencing (Applied Biosystems, Foster City, CA) using the manufacturer's instructions.
All doxycycline-inducible plasmids were purified by Qiagen preparation (Qiagen Sciences) and transfected into H1299 cells with calcium phosphate and 2x DNA precipitation buffer (5-Prime 3-Prime, Boulder, CO). Stable clones were selected with 200 µg/ml of hygromycin (Invitrogen). We initially selected clones with inducible expression of EGFP by treating cells with 2 µg/ml of doxycycline (Sigma, St. Louis, MO) and screening for green fluorescence using an Olympus IX50 inverted epifluorescent microscope (Olympus, Melville, NY) equipped with a Dage RC300 camera (Dage RC300; Dage-MTI, Michigan City, IN) and computer with image-capturing software (Scion Imager V3.0; Scion, Frederick, MD). Fluorescent colonies were picked and propagated in the absence of doxycycline.
p53 transcriptional activity. We plated cells in triplicate at 300,000 cells/60-mm dish by culturing them overnight. Each plate was transfected with 2 µg of p53-luciferase plasmid (Stratagene, La Jolla, CA) and 0.5 µg of the Renilla luciferase-expressing plasmid pRL-SV40 (Promega, Madison, WI) by calcium phosphate precipitation (5-Prime 3-Prime). Transfected cells were harvested 1 day later, and luciferase activity was determined with the dual luciferase reporter assay system (Promega). Relative luciferase activity from the p53-luciferase plasmid was normalized to Renilla luciferase.
RNA extraction and analysis. Cells were harvested in 4 M guanidine isothiocyanate, 0.5% N-laurylsarcosine, 20 mM sodium citrate, and 0.1 M 2-mercaptoethanol. RNA was extracted using acid phenol and phase lock gel columns (5-Prime 3-Prime) and resuspended in diethylpyrocarbonate-treated water. The amount of RNA in an aqueous solution was determined by absorbance at 260 nm. RNase protection assays were performed with the human cell cycle-2 multiprobe template kit according to the manufacturer's instructions (PharMingen, San Diego, CA) as described (15). Protected products were separated on a 6% acrylamide-8 M urea sequencing gel, dried, and visualized by exposure on PhosphorImager screens. Band intensities were quantified with ImageQuant software (Molecular Dynamics, Sunnyvale, CA) and normalized to expression of L32.
Western blot analysis. Cells were lysed in 50 mM Tris (pH 8.0), 120 mM NaCl, and 0.5% Nonidet P-40 supplemented with 2 mg/ml of aprotinin and 100 mg/ml of phenylmethylsulfonyl fluoride. The lysate was cleared by centrifugation and protein concentrations determined by the Lowry assay (DC Protein Assay; Bio-Rad, Hercules, CA). The lysates were boiled for 5 min in 3x Laemmli buffer (1x Laemmli contains 50 mM Tris, pH 6.8, 1% 2-mercaptoethanol, 2% SDS, 0.1% bromphenol blue, and 10% glycerol). Proteins were separated by SDS-15% polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA). The membranes were then incubated in primary antibodies for p21 (1:500, PharMingen), p27 (1:100; Santa Cruz Biotechnology, Santa Cruz, CA), EGFP (1:1,000, Clontech), FLAG (1:250, Sigma), or, as a loading control, -actin (1:5,000, Sigma). Antibodies were diluted in Tris-buffered saline plus 5% Tween 20 (TBST) or TBST plus 5% milk per manufacturer's instructions. Membranes were then incubated in the appropriate secondary antibody in TBST plus 5% milk (goat anti-mouse, Southern Biotechnology, Birmingham, AL; rabbit anti-goat or goat anti-rabbit, Jackson Labs, West Grove, PA). We visualized specific antibody interactions by chemiluminescence using ECL plus Western Blotting Detection System (Amersham, Arlington Heights, IL).
Flow cytometry. Cells were trypsinized, resuspended in their original medium, and centrifuged at 300 g. The medium was removed, and the cells were fixed in 75% ethanol for 24 h. The cells were resuspended in 1 ml of RNase (1 mg/ml) for 30 min, centrifuged, and resuspended in 0.5 ml of propidium iodide (10 µg/ml) dissolved in phosphate-buffered saline. The samples were analyzed on an Epics Elite ESP (Coulter Electronics, Hialeah, FL) flow cytometer to collect 10,000 events. Percentages of cells in G1, S, and G2/M were determined by use of EPICS CytoLogic Software (Coulter).
Statistical analysis. Values are means ± SD. Group means were compared by ANOVA using Fisher's procedure post hoc analysis with StatView (Abacus Concepts, Berkeley, CA) software for Macintosh. P < 0.05 was considered significant.
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RESULTS |
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p53 restores p21 induction during hyperoxia in H1299 cells. Parental H1299 and H1299+p53 were transiently transfected with a p53-luciferase plasmid containing 15 copies of the p53-binding site within the p21 promoter. Luciferase activity was measured as an indicator that p53 transcriptional activity had been restored. Compared with the parental p53-deficient H1299 cells, which exhibited low luciferase activity, H1299+p53 cells exhibited 30- to 50-fold more activity, indicating that p53 transcription had been restored (Fig. 2A). This high p53 transcriptional activity in the absence of genotoxic stress is consistent with previous studies showing that the p53-dependent DNA damage response becomes activated when plasmid DNA is transfected into cells (28).
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RNase protection assays were used to investigate p53 and p21 mRNA expression (Fig. 2B). As expected, p53 mRNA was not detected in p53-deficient H1299 cells. Although p21 mRNA was detected, it did not increase during hyperoxia. Interestingly, expression of other members of the Cip (p27 and p57) and INK4 (p15, p16, p18, and p19) families also did not change during hyperoxia. Similarly, hyperoxia did not alter expression of the pocket proteins retinoblastoma (Rb), p107, or p130. In contrast, high constitutive levels of p53 mRNA were readily detected in H1299+p53 cells, which remained constant during hyperoxia. Restoration of p53 in H1299+p53 cells resulted in a three- to sevenfold induction of p21 mRNA after 2 and 3 days of hyperoxia (Fig. 2C). This was considerably slower than that reported in A549 and HCT116 cell lines, where p21 increased within the first 24 h of exposure (15, 27). Expression of p27 (Fig. 2D), p57, all four members of the INK4 family, or the three Rb pocket proteins was not significantly affected by p53.
Flow cytometry was used to determine whether restoration of p53 enhanced the proportion of H1299+p53 cells in G1 during hyperoxia (Fig. 3). G1 DNA content was observed in 45% of parental H1299 and H1299+p53 cells exposed to room air. Three days of hyperoxia markedly reduced the proportion of H1299 cells in G1 to
10%. This was associated with an increase in the proportion of cells in S and G2. Although hyperoxia also reduced the proportion of H1299+p53 cells in G1, approximately twofold more cells remained in G1 compared with the parental H1299 cells. Although the proportion of cells in G1 was not significantly different between the two cell lines (P = 0.06), a definite trend toward more H1299+p53 cells in G1 was observed. One explanation may be that p21 did not increase until 48 h, by which time cells were arresting in S and G2. Together, these findings confirm that hyperoxia induces p53-dependent induction of p21, which is associated with greater retention of cells in G1.
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p21 and p27 exert G1 arrest during hyperoxia. Although p53 restored induction of p21 and G1 arrest during hyperoxia, it remains possible that G1 arrest depended on other factors besides p21. Regulated overexpression of EGFP, EGFp21, or EGFp27 in parental H1299 cells was used to test this hypothesis. Because glutathione S-transferase or histidine tags added to the amino terminus of p21 did not affect interactions with Cdk or PCNA (14, 29, 36), EGFP was placed in frame with p21 and p27 to allow recombinant proteins to be visualized during exposure. Western blot analysis using antibodies against EGFP, p21, or p27 confirmed that the recombinant proteins were highly expressed when cells were treated with doxycycline (Fig. 4). Some proteolysis of EGFP and EGFp21 proteins was observed when blots were probed with anti-EGFP serum. EGFP, but not EGFp21 or EGFp27, was faintly detected when blots were intentionally overexposed (data not shown). Because leaky expression of kinase inhibitors would reduce growth, this implies that tightly regulated expression of p21 and p27 had been achieved.
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Intracellular localization was assessed by intrinsic green fluorescence afforded by EGFP. In cells expressing EGFP, faint cytoplasmic fluorescence was detected in the absence of doxycycline that markedly increased when doxycycline was added (Fig. 5). This low fluorescence in untreated cultures confirms the low level of protein detected when Western blots were overexposed. In contrast, intense nuclear fluorescence was detected when doxycycline was added to induce EGFp21 or EGFp27. Nuclear localization of the fusion proteins is attributed to the nuclear localization sequence present in the carboxy terminus of p21 and p27.
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Flow cytometry was used to determine whether recombinant proteins were capable of altering cell cycle progression. We cultured cells in the absence or presence of doxycycline for 1 day before exposing them to room air or hyperoxia for an additional 2 days. EGFP, EGFp21, and EGFp27 cells cultured in room air with or without doxycycline displayed a similar DNA profile with the majority of cells exhibiting G1 DNA content (Fig. 6). Closer examination indicated that more EGFp27 cells were in G1 when cells were treated with doxycycline. As expected, in the absence of doxycycline, hyperoxia markedly decreased the proportion of cells in G1 to 10% while increasing the proportion in S and G2. In contrast, the proportion of cells in G1 remained significantly higher when EGFp21 (P < 0.005) or EGFp27 (P < 0.0001) was induced by doxycycline. In fact, an even larger proportion of cells expressing EGFp27 were retained in G1 during hyperoxia compared with cells expressing EGFp21. In contrast, induction of EGFP by itself did not prevent the dramatic depletion of cells in G1 (P = 0.29). Thus overexpression of EGFp21 or EGFp27 during hyperoxia enhanced the proportion of cells arrested in G1.
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Both Cdk and PCNA binding domains of p21 inhibit proliferation during hyperoxia. Previous studies showed that the amino-terminal Cdk and carboxy-terminal PCNA binding domains inhibit DNA synthesis when individually expressed in cells, albeit not as robustly as full-length protein (19, 29). Although the effect of EGFp21 and EGFp27 suggests that the common Cdk domain is functional during hyperoxia, the PCNA binding domain may not be active because functional PCNA is anticipated to be required for DNA repair. Regulated overexpression of amino-terminal and carboxy-terminal (p21Amino and p21Carboxy, respectively) p21 domains in parental H1299 cells was used to test this hypothesis. Western blot analysis using anti-Flag antiserum confirmed that doxycycline induced expression of individual p21 domains of 11 kDa (Fig. 7). Some expression of the p21Amino domain was observed in the absence of doxycycline. Doxycycline also increased EGFP translated off the downstream ORF. Equal levels of
-actin confirmed that all lanes contained equivalent amounts of protein.
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Flow cytometry was used to investigate whether individual domains of p21 were capable of exerting G1 arrest during hyperoxia. p21Amino and p21Carboxy cells were cultured in the absence or presence of doxycycline for 1 day before being exposed to hyperoxia for an additional 2 days. DNA histograms of both cell lines exposed to room air in the absence or presence of doxycycline were indistinguishable (Fig. 8). In the absence of doxycycline, hyperoxia reduced the proportion of cells in G1 and increased the proportion in S and G2. In contrast, the proportion of cells in G1 was significantly higher when p21Amino (P < 0.005) and p21Carboxy (P < 0.0005) domains were induced by doxycycline. Overexpression of p21Carboxy protein resulted in twice as many cells remaining in G1 as overexpression of p21Amino protein. This occurred even though more p21Amino protein was detected by Western blot analysis, suggesting that the p21Carboxy is the more potent inhibitory domain.
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DISCUSSION |
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An important finding in the current study is that both domains on p21 remain functionally capable of exerting G1 arrest even when oxidative damage that might inactivate proteins is occurring. The determination that the amino-terminal Cdk domain exerts G1 arrest is consistent with the observation that hyperoxia decreased cyclin E-dependent kinase activity in SV40-T2 cells (8). Indeed, overexpression of EGFp27, which contains a homologous amino-terminal Cdk domain, also exerted G1 arrest during hyperoxia. Interestingly, the proportion of EGFp27 cells retained in G1 was significantly greater than EGFp21. On the basis of in vitro kinase assays, p27 has greater activity toward cyclin E-Cdk2 than p21 (14, 25). However, p21 has higher activity toward S phase cyclin A-Cdk2 than p27. Because p27 has greater activity toward early G1 kinases than p21, overexpression of EGFp27 is expected to be more effective at preventing S phase entry than EGFp21. Alternatively, p27 may be more effective in this model because it is more stable than p21. Because such supraphysiological levels of p21 and p27 are expressed in this system, it is difficult to reconcile whether varied expression levels are responsible for the observed differences in the proportion of cells arrested in G1. p27 mediates G1 arrest of Mv1Lu cells grown to confluence or treated with TGF- (24). Like the parental H1299 cells in this study, Mv1Lu epithelial cells growth arrest in S phase during hyperoxia because they fail to express p21 (27). In contrast, cells could be maintained in G1 by growing to confluence or treating with TGF-
, pathways that activate p27 (26). These findings reveal that blocking Cdk activity is one mechanism by which cells can arrest in G1 during hyperoxia.
An additional finding was that the carboxy-terminal PCNA binding domain also exerts G1 arrest during hyperoxia. PCNA is a homotrimer that provides a scaffold for attachment of DNA polymerases and other factors required for replication and repair (17). A synthetic peptide derived from the carboxy terminus of p21 bound PCNA and reduced DNA polymerase--catalyzed chain elongation (13, 36). Transient overexpression of the PCNA binding domain in R1B and U2OS cells inhibited [3H]thymidine and BrdU incorporation (19, 29). In those studies, the p21 PCNA binding domain was less efficient at inhibiting DNA synthesis than the Cdk domain, which was also less efficient than the intact protein. Increased activity of the Cdk-binding domain relative to the PCNA-binding domain was argued to be a reflection of its greater stability in U2OS cells. In the current study, the PCNA domain exerted more G1 arrest during hyperoxia than the Cdk domain, even though less protein was expressed. This suggests that G1 arrest during hyperoxia may be caused predominantly by p21-PCNA interactions rather than p21-Cdk interactions. This hypothesis is currently under investigation.
p21 may participate in long-patch base excision repair or nucleotide excision repair (NER) through its interactions with PCNA, a component of both processes. However, it remains controversial whether p21 aids or represses repair. For example, it is well established that p21-deficient mice and cell lines are acutely sensitive to DNA-damaging agents such as ionizing radiation, cisplatin, nitrogen mustard, UV (10, 12, 20, 31), and hyperoxia (15, 23). In addition to increased survival, cells expressing p21 had enhanced ability to repair plasmid reporters that were damaged by alkylation or UV irradiation. Although p21 blocks DNA replication, it does not block in vitro repair of damaged oligonucleotides (18, 34). Other studies, however, have argued that p21 inhibits repair. For example, overexpression of p21 in HT1080 fibrosarcoma cells reduced expression of several DNA repair enzymes involved in NER (5). Similarly, the PCNA binding domain of p21 inhibited NER activity in vitro and in cultured cells (7, 34). We are currently investigating how p21-PCNA interactions might affect repair of oxidized DNA.
Our current study also shows that mRNA expression of the Cip/Kip, INK4, and Rb protein families was unaltered by hyperoxia when p53 was absent. Although expression remained constant when p53 was restored, p21 became inducible by hyperoxia. Induction of p21 was slower by 24 h compared with A549 and HCT116 cell lines, which may explain why the G1 delay shown in Fig. 3 was modest (15, 27). Nonetheless, p21 expression was dependent on p53. Similar findings were observed in HCT116 cells that express wild-type p53 and an isogenic line in which p53 was deleted (15). In those cells, hyperoxia induced p53-dependent expression of p21 without affecting expression of other Cip, INK4, or Rb proteins. These findings are distinctly different from that reported for SV40-T2 cells that showed hyperoxia increased p21 and p27 mRNAs (8). Because SV40 large T antigen used to immortalize the cells disrupts p53 activity, it is possible that other signal transduction pathways are also affected to allow induction of p21 and p27 during hyperoxia. This could explain why TGF- signaling was suggested to participate in hyperoxia-induced growth arrest of SV40-T2 cells, while not being required in the H1299 cell line used in the current study (4).
In summary, the current study established that in vitro exposure to hyperoxia induces p53-dependent expression of p21 that exerts G1 arrest through both Cdk and PCNA binding domains. Cells that fail to express p21 arrest in S and G2 through other mechanisms that remain to be characterized. A significant finding was that both Cdk and PCNA binding domains on p21 remained growth inhibitory even under oxidative stress. The generation of cell lines with regulated expression of these domains will be useful for clarifying how p21 integrates DNA replication with repair.
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ACKNOWLEDGMENTS |
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GRANTS
This work was funded in part by National Heart, Lung, and Blood Institute Grants HL-58774 and HL-67392 (M. A. O'Reilly). National Institutes of Health training Grants ES-07026 and HL-66988 supported C. E. Helt. The flow cytometry facility is supported in part by National Institute of Environmental Health Sciences Center Grant ES-01247.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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