Affiliations of authors: A. Fernandez, T. Udagawa, C. Schwesinger, W.-D. Beecken, E. Achilles-Gerte, Department of Surgery, Division of Surgical Research, Children's Hospital, Boston, MA; T. J. McDonnell, Department of Molecular Pathology, The University of Texas M. D. Anderson Cancer Center, Houston; R. J. D'Amato, Department of Surgery, Division of Surgical Research, Children's Hospital, Boston, and Department of Ophthalmology, Harvard Medical School, Boston.
Correspondence to: Robert J. D'Amato, M.D., Ph.D., Department of Surgery, Division of Surgical Research, Children's Hospital, 300 Longwood Ave., Boston, MA 02115 (e-mail: Robert.damato{at}tch.harvard.edu).
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ABSTRACT |
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INTRODUCTION |
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Through the stimulation of angiogenesis and the inhibition of apoptosis, tumors try to escape the effects of hypoxia. Hypoxia becomes an increasing problem for rapidly growing tumors for two reasons. First, tumor cells expand beyond the limits of oxygen diffusion from existing blood vessels (5). Second, although hypoxia induces tumors to express angiogenic growth factors during the angiogenic switch, the new blood vessels that result are immature, with leaky capillaries and abnormal blood flow (6). The persistence of hypoxic conditions, therefore, frequently results in the induction of apoptosis (7). However, hypoxic stress can also exert a selective pressure within tumors that supports the expansion of cells with mutations that reduce the apoptotic potential of those cells (8).
Much is known about the genetic changes in tumors that result in the selection of mutant cells with decreased apoptotic potential. These changes include the inactivation of proapoptotic genes, such as p53 (also known as TP53) and BAX, and overexpression of antiapoptotic genes, such as bcl-2 (912). The bcl-2 gene was first identified in association with the t(14;18) chromosomal translocation in follicular lymphomas (13). Overexpression of bcl-2 inhibits apoptosis and is associated with malignant progression of tumors of epithelial origin (12). Transfection of bcl-2 into tumor cell lines increases their tumorigenicity in vivo, resulting in cancers that are resistant to treatment with conventional therapies (14). The oncogenic properties of bcl-2 have been attributed mainly to its ability to inhibit apoptosis (15,16). Several mechanisms have been proposed to explain this activity of bcl-2, including regulation of mitochondrial permeability and release of proapoptotic proteins from mitochondria (17,18), regulation of p53 transport across the nuclear membrane (19), and regulation of glutathione redox systems (20).
Because bcl-2 overexpression by tumors promotes their survival by decreasing apoptosis in the setting of hypoxia-induced stress, we hypothesized that bcl-2 might also enhance tumor survival in hypoxia by further stimulating angiogenesis. The effect of bcl-2 on angiogenesis could be through direct or indirect pathways. bcl-2 may, for example, stimulate angiogenesis indirectly by increasing resistance to hypoxia-induced apoptosis so that the lifespan of the tumor cells is lengthened and the secretion of angiogenic factors is prolonged. Alternatively, bcl-2 could act directly (and independently of its effects on apoptosis) to increase the overall production of angiogenic factors in response to hypoxia. Because bcl-2 has been shown to be an important factor regulating the malignant progression of prostate carcinoma (12), we tested this hypothesis by determining the effect of bcl-2 overexpression on the angiogenic potential of the PC3, LNCaP, and DU-145 prostate carcinoma cell lines.
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MATERIALS AND METHODS |
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Human prostate carcinoma cell lines PC3, LNCaP, and DU-145 were stably transfected with a spleen focus-forming virus-based expression vector that either contained or lacked the complementary DNA (cDNA) encoding human bcl-2, as described previously (21,22). Single-cell clones were selected after growing the transfected cells in G418 to select for the neomycin (neo) resistance gene contained on the vector and were expanded in culture to generate clonal populations of cells. Cells were cultured in Dulbecco's modified Eagle medium (JRH Biosciences, Lenexa, KS) and were supplemented with 10% fetal bovine serum, 0.29 mg/mL glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin in a humidified 10% CO2 incubator.
Cell viability measurements were performed before injection in the tumor growth experiments. The number of viable cells was determined by use of a hemocytometer to count trypsinized cells that excluded the vital dye trypan blue.
Cell Incubation Under Normoxic and Hypoxic Conditions
Cells were incubated in hypoxic (10% CO2, 1% O2, and 89% N2) or normoxic (10% CO2 and 19% O2) conditions for various times. Oxygen concentrations were maintained at the desired levels by injecting the appropriate amounts of nitrogen gas into the incubators.
Enzyme-Linked Immunosorbent Assay for Vascular Endothelial Growth Factor Analysis
Conditioned media were collected from cells grown in culture, and, following centrifugation at 4000g for 5 minutes at 4 °C, the supernatants were frozen at -20 °C. The levels of vascular endothelial growth factor (VEGF) protein were measured in conditioned media samples by use of the Quantikine ELISA kit (R&D Systems, Minneapolis, MN) according to the manufacturer's instructions.
Measurement of Apoptosis
Cells were washed twice with phosphate-buffered saline (PBS), treated with trypsin, and washed twice more with PBS. After the washes, the extent of apoptosis was determined by measuring Annexin V-fluorescein isothiocyanate binding to the cells by fluorescence-activated cell-sorting analysis by use of an Annexin V detection kit from Pharmingen (San Diego, CA), according to the manufacturer's instructions.
Western Blotting for Detection of Hypoxia-Induced Transcription Factor HIF-1
Cell lysates were prepared by adding 100 µL lysis buffer (i.e., 120 mM NaCl, 50 mM Tris, and 0.5% Nonidet P-40) to 0.5 x 106 cells and incubating them on ice for 20 minutes. Protein concentration was determined by the Bio-Rad DC Protein assay (Bio-Rad Laboratories, Hercules CA). Protein samples (50 µg) were boiled in sodium dodecyl sulfate (SDS) gel-loading sample buffer (i.e., 50 mM TrisHCl [pH 6.8], 100 mM dithiothreitol, 2% SDS, 0.1% bromophenol blue, and 10% glycerol), separated on 8% SDSpolyacrylamide gels, and transferred to nitrocellulose membranes (Biorad, Melville NY). Nitrocellulose membranes were blocked in PBS containing 0.05% Tween 20 and 3% nonfat dry milk and then incubated with a mouse monoclonal anti-HIF-1 antibody (Affinity Bioreagents, Inc., Golden, CO) diluted 1 : 2000. Following three 15-minute washes in PBS with 0.05% Tween 20, blots were incubated with horseradishperoxidase-conjugated rabbit anti-rat antibody at a 1 : 10 000 dilution. Bands were visualized by chemiluminescence, and relative levels of HIF-1
were determined by densitometric scanning and normalization against an actin control.
Xenograft Tumor Growth
Prostate carcinoma cells stably transfected with either empty vector (designated by the suffix, "-neo") or vector containing the bcl-2 cDNA (designated by the suffix, "-bcl-2") were injected in the dorsal subcutaneous space of severe combined immunodeficiency (SCID) mice at a dose of 106 cells per mouse, and subsequent tumor growth was assessed twice a week. When tumors reached approximately 100 mm3, mice were randomly assigned to control (four mice) and experimental (five mice) groups. Mice in the experimental group received therapy with the angiogenesis inhibitor TNP-470 (30 mg/kg every other day by subcutaneous injection) or the cytotoxic drug mitomycin C (1 mg/kg once a week by intraperitoneal injection). The width (W) and length (L) of each tumor was measured biweekly on the mice with calipers. Tumor volume was calculated according to the formula L x W2 x 0.52. All animal studies have been approved by the animal use committee at Children's Hospital and adhered to institutional guidelines for animal care.
Mouse Corneal Tumor Angiogenesis Assay
The procedure is a modification of that described by Muthukkaruppan et al. (23). Briefly, mice were anesthetized by intraperitoneal injection of 750 mg/kg Avertin (Aldrich, St. Louis, MO) and topical application of proparacaine HCl (Allergan, Irvine, CA). An intrastromal linear keratotomy was made by use of a number 10 surgical blade angled at 30 degrees. A corneal micropocket was dissected toward the temporal limbus by use of a modified von Graefe knife #3 (2 x 30 mm). Untreated, subcutaneous xenograft tumors derived from injections of PC3-neo and PC3-bcl-2 cells were excised from carrier mice, and a fragment of approximately 0.3 x 0.3 x 0.3 mm was removed from the tumor while it was viewed under a dissecting microscope. This fragment was placed in the corneal micropocket and advanced to 1 mm from the limbus by use of the von Graefe knife. Topical erythromycin was applied once to the eye that had surgery. Neovascularization of the tumor fragment within the eye that had surgery was assessed at weekly intervals by use of a slit lamp for illumination and magnification.
Immunohistochemical Analysis of CD31 Expression
Sections from paraffin-embedded tumors derived from prostate carcinoma-bcl-2 or -neo cells were incubated overnight with the rat anti-mouse CD31 (an endothelial cell-specific antigen) monoclonal antibody (Pharmingen, San Diego, CA) in TNB (i.e., 0.1 M TrisHCl [pH 7.5], 0.15 M NaCl, and 0.5% blocking reagent from the tyramide signal amplification (TSA) indirect amplification kit [Du Pont NEN, Boston, MA]) plus 10% rabbit serum. After incubation with a rabbit anti-rat secondary antibody (Vector Laboratories, Inc., Burlingame, CA), the antibodyantigen complexes were visualized by use of a TSA indirect amplification kit and the Vectastain ABC-AP kit (Vector Laboratories, Inc.). Stained tumor sections were scanned at low magnification to identify areas of high vessel density. For each section, CD31-positive cells within three areas of high vessel density were counted while viewed at high magnification (x100). From each group of tumors (-neo and -bcl-2), two tumors were selected and the number of stained cells on five sections were counted from each one.
Statistical Analysis
P values for the enzyme-linked immunosorbent assay experiments described in the text and in the legend for Fig. 1 were determined by use of a two-tailed Student's t test of data collected from two independent experiments performed in triplicate. For the tumor experiments, the volumes determined from measurements taken 1 day after the final treatment were analyzed by a two-tailed Student's t test. All P values are two-sided.
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RESULTS |
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To examine the effect of bcl-2 overexpression on VEGF production, three different prostate carcinoma cell lines cells were stably transfected with vector only or with vector bearing the bcl-2 cDNA. Control PC3 transfectants (PC3-neo) and PC3 cells overexpressing bcl-2 (10-fold increase in bcl-2 protein compared with vector control; data not shown) were cultured in either normoxic (19% O2) or hypoxic (1% O2) conditions, and the VEGF protein levels were measured in the conditioned media. After 72 hours of culture in normoxic conditions, there was a small but statistically significant increase (P = .04) in the amount of VEGF secreted by PC3-bcl-2 cells (32 pg/104 cells; 95% confidence interval [CI] = 31 to 33 pg/104 cells) relative to that secreted by PC3-neo cells (20 pg/104 cells; 95% CI = 19.2 to 20.8 pg/104 cells) (Fig. 1, A). However, when cells were cultured in hypoxic conditions, there was a larger (2.3-fold) increase in the amount of VEGF secreted by the PC3-bcl-2 cells (91.5 pg/104 cells; 95% CI = 86 to 96 pg/104 cells) compared with that secreted by the PC3-neo cells (38.9 pg/104 cells; 95% CI = 37 to 41 pg/104 cells) (Fig. 1
, A). The difference in the amount of VEGF secreted by PC3-neo and PC3-bcl-2 cells cultured in hypoxic conditions was evident by 24 hours and was pronounced at 48 and 72 hours (P = .001). Culturing in hypoxic conditions did not affect either the very low endogenous levels of bcl-2 in the PC3-neo cells or the 10-fold higher levels of bcl-2 in the PC3-bcl-2 cells (data not shown). Analysis of additional clones of PC3-bcl-2 cells revealed a direct association between the level of bcl-2 expression and the amount of VEGF secreted in both normoxic and hypoxic conditions.
A similar association between bcl-2 expression and VEGF secretion was detected in LNCaP cells that were stably transfected with vector only (LNCaP-neo) or with the bcl-2 cDNA (LNCaP-bcl-2) and cultured in hypoxic conditions. LNCaP-neo and LNCaP-bcl-2 cells secreted similar levels of VEGF when cultured for 48 hours in normoxic conditions (30 and 24 pg/104 cells; 95% CI = 24 to 36 pg/104 cells and 14 to 34 pg/104 cells, respectively). After 48 hours of exposure to hypoxic conditions, LNCaP-bcl-2 cells secreted statistically significantly (P = .04) more VEGF (106 pg/104 cells; 95% CI = 98 to 114 pg/104 cells) than did LNCaP-neo cells (63 pg/104 cells; 95% CI = 52 to 74 pg/104 cells).
By contrast, analysis of DU-145 cells stably transfected with vector (DU-145-neo) or with the bcl-2 cDNA (DU-145-bcl-2) revealed that DU-145-bcl-2 transfectants secreted statistically significantly (P = .001) higher levels of VEGF in normoxic conditions (226 pg/104 cells; 95% CI = 218 to 234 pg/104 cells at 24 hours) than did DU-145-neo transfectants (106 pg/104 cells; 95% CI = 102 to 110 pg/104 cells ). Culture in hypoxic conditions resulted in the rapid death of DU-145-neo cells, thus preventing the analysis of their VEGF secretion behavior under hypoxia. It is interesting to note that, in normoxic conditions, the three cell lines responded differently to bcl-2 overexpression: bcl-2 overexpression caused a strong increase in VEGF secretion in the DU-145 cells, caused a moderate increase in VEGF secretion in PC3 cells, and had no effect on VEGF secretion in LNCaP cells. These observations suggest that bcl-2 interacts with another factor or factors to regulate VEGF secretion, and that these factors are expressed at different constitutive levels in different tumor cells.
To establish whether bcl-2 overexpression could affect the secretion of angiogenic factors other than VEGF, we measured the levels of basic fibroblast growth factor (bFGF) in the culture media of stably transfected PC3 cells that were exposed to normoxic and hypoxic culture conditions (Fig. 1, B). Overexpression of bcl-2 resulted in no differences in the amount of bFGF secreted by the bcl-2 versus control cell lines in normoxia (P = .4) or hypoxia (P = .3). These data suggest that the effects of bcl-2 on VEGF secretion are relatively specific.
To determine whether bcl-2 had an indirect effect on VEGF secretion under hypoxic conditions, that is, if the secretion was due to enhanced cell survival resulting from increased resistance to hypoxia-induced apoptosis, we measured annexin V staining to determine the extent of hypoxia-induced apoptosis in PC3-neo and PC3-bcl-2 cells. After 72 hours of incubation in hypoxic conditions, neither cell line showed an increase in apoptosis compared with the cells grown in normoxic conditions (16.9% versus 15.8% in PC3-neo cells and 9.1% versus 9.7% in PC3-bcl-2 cells; Fig. 2). These data demonstrate that PC3 cells are inherently resistant to hypoxia-induced apoptosis, regardless of their bcl-2 expression status, implying that bcl-2 expression does not have an indirect effect on hypoxia-induced secretion of VEGF by enhancing cell survival. To demonstrate that bcl-2 was functional in the PC3-bcl-2 cells, we stressed the cells by growing them in the absence of serum. This treatment resulted in a marked increase in apoptosis in the PC3-neo cells (48.4%) but not in the PC3-bcl-2 cells (15%) (Fig. 2
). Thus, overexpression of bcl-2 in the PC3-bcl-2 cells inhibits apoptosis triggered by stimuli other than hypoxia.
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HIF-1 Expression in PC3-bcl-2 and PC3-neo Cells
Induction of VEGF expression in response to hypoxia requires the activation of the transcription factor HIF-1 (24,25). To determine whether the differences in VEGF expression detected between the PC3-derived cell lines were mediated by the induction of the expression of HIF-1
, we used western blotting to examine the levels of HIF-1
in cells that were cultured in normoxic and hypoxic conditions. HIF-1
was expressed at similar levels in the PC3-bcl-2 and the PC3-neo cells cultured in normoxic conditions (data not shown). Exposure to hypoxic conditions resulted in a similar induction of HIF-1
in PC3-neo (2.4-fold) and PC3bcl-2 (2.2-fold) cells (data not shown). This observation suggests that a mechanism other than the regulation of the levels of HIF-1
is responsible for the stimulatory effects of bcl-2 on VEGF secretion in PC3 cells under hypoxic conditions.
Tumor Growth and Vessel Density of bcl-2 Overexpressing Xenografts
To test whether the enhanced expression of VEGF in response to hypoxia in the bcl-2 overexpressing cells in vitro would result in higher levels of neovascular activity in vivo, we inoculated PC3-neo and PC3-bcl-2 cells subcutaneously into SCID mice. Tumors derived from PC3-bcl-2 cells grew at approximately twice the rate of tumors derived from PC3-neo cells (Fig. 3). At day 37 after inoculation, tumors were removed and analyzed for microvessel density by measuring the expression of the endothelial cell-specific marker CD31. The PC3-bcl-2-derived tumors had, on average, twice as many CD31-positive cells than the PC3-neo-derived tumors (134 cells [95% CI = 118 to 150 cells] versus 57 cells [95% CI = 49 to 65 cells] per x100 field, respectively; Fig. 4
, A and B). Immunohistochemical analysis of tumors derived from DU-145-bcl-2 and DU-145-neo cells revealed a similar difference in the number of CD31-positive cells (76 CD31-positive cells [95% CI = 66 to 86 CD31-positive cells] and 34 CD31-positive cells [95% CI = 28 to 40 CD31-positive cells] per x100 field, respectively; Fig. 4
, C and D).
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Induction of Angiogenesis in bcl-2-Overexpressing Tumors Implanted in a Cornea Micropocket
To determine the angiogenic potential of PC3-neo and PC3-bcl-2 cells, we measured the ability of tumors derived from each cell line to induce neovascularization after implantation into corneal micropockets. One week after tumor implantation, corneal angiogenesis (i.e., as assayed by the presence of vessels at least 1 mm long sprouting from the limbus vessel into the cornea) was detected in six of the eight corneas implanted with tumors derived from PC3-bcl-2 cells. In that same time period, none of the eight corneas implanted with tumors derived from PC3-neo cells displayed corneal angiogenesis. At 5 weeks after tumor implantation, vessels were detectable in 25% of the corneas implanted with tumors derived from PC3-neo cells. Whereas tumors derived from both the PC3-neo and PC3-bcl-2 cells induced morphologically similar vessels in this assay, tumors derived from the PC3-bcl-2 cells did so at a faster rate. Thus, the differences in induced neovascularization were due to the stronger angiogenic potential of the PC3-bcl-2 tumors and not to decreased viability of the PC3-neo-derived cells.
Treatment of bcl-2-Overexpressing Tumors With Antiangiogenic Therapy and Conventional Chemotherapy
Because tumors derived from PC3-bcl-2 cells stimulated angiogenesis, we examined whether angiogenesis inhibitors could effectively treat bcl-2-overexpressing tumors. PC3-neo and PC3-bcl-2 cells were injected into SCID mice to induce tumor formation. Once the tumors reached a volume of 100 mm3, the mice were treated with the antiangiogenic drug TNP-470. As shown in Fig. 3, A, TNP-470 strongly inhibited the growth of tumors derived from both PC3-bcl-2 and PC3-neo cells. The untreated PC3-bcl-2-derived tumors grew, on average, to a final volume roughly twice that of the untreated PC3-neo-derived tumors (1301 mm3 [95% CI = 1158 to 1543 mm3] and 587 mm3 [95% CI = 520 to 654 mm3, respectively]; P = .03). However, treatment with TNP-470 reduced the final volume of both types of tumors to a similar level (206 mm3 [95% CI = 173 to 239 mm3; P = .04] for PC3-bcl-2-derived tumors and 191 mm3 [95% CI = 174 to 208 mm3; P = .01] for PC3-neo-derived tumors). TNP-470 was also effective in decreasing the mean volume of tumors derived from both DU-145-bcl-2 cells (from 1043 mm3 [95% CI = 930 to 1158 mm3] for untreated tumors to 271 mm3 [95% CI = 187 to 355 mm3; P = .004] for treated tumors) and DU-145-neo cells (from 439 mm3 [95% CI = 320 to 568 mm3] for untreated tumors to 253 mm3 [95% CI = 146 to 360 mm3; P = .05] for treated tumors).
In contrast to their sensitivity to the antiangiogenic treatment, tumors dervived from PC3-bcl-2 cells were completely resistant to the chemotherapeutic agent mitomycin C (the mean volume of untreated tumors was 950 mm3 [95% CI = 853 to 1047 mm3], while that of treated tumors was 997 mm3 [95% CI = 878 to 1116 mm3; P = .7]). PC3-neo-derived tumors, by contrast, responded to mitomycin C treatment (the mean volume of untreated tumors was 687 mm3 [95% CI = 523 to 851 mm3], while that of treated tumors was 81 mm3 [95% CI = 67 to 97 mm3; P = .05]; Fig. 3, B). This result is consistent with our observation that PC3-bcl-2 cells are 2.2 times as resistant to treatment with mitomycin C in vitro as PC3-neo cells (data not shown). This observation raises the possibility that apoptosis-resistant tumor cells, such as PC3-bcl-2, could give rise to tumors that are resistant to treatment with apoptosis-inducing agents while still being responsive to angiogenesis inhibitors, which target the endothelial cells in the tumor vasculature.
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DISCUSSION |
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The regulation of VEGF secretion by bcl-2 is a new function for this oncogene, with important implications concerning bcl-2's ability to support tumor progression. Critical steps in tumor progression include increased proliferation, induction of angiogenesis, inhibition of apoptosis, activation of telomerase, and independence from growth and survival factors (1). In this context, single genetic alterations that have the ability to confer several of these changes will have stronger effects on tumor progression than will those that affect only one step. Genes that are altered in such ways could thus be considered as more effective oncogenes than those with alterations that affect only one of these processes. Our results suggest that bcl-2 is an oncogene that has the ability to contribute to the tumor progression process at multiple levels, that is, through both the inhibition of apoptosis and the induction of angiogenesis.
It is interesting to note that the overexpression of bcl-2 caused an increase in VEGF secretion in PC3 and DU-145 cells that were cultured in normoxic conditions but not in LNCaP cells cultured in normoxic conditions. Because bcl-2 was expressed at comparable levels in these three cell lines, these results suggest that the effect of bcl-2 on VEGF expression may require a factor (or factors) that is expressed constitutively in some cell lines (PC3 and DU-145), but only in response to hypoxia in others (LNCaP).
Tumors derived from cells that overexpress bcl-2 grow more aggressively in vivo than tumors derived from cells that do not overexpress bcl-2. This phenomenon has been attributed to the antiapoptotic properties of bcl-2 (26). However, our observations suggest that bcl-2-overexpressing tumors may grow faster because they also induce more neovascularization. This possibility is supported by our TNP-470 treatment data. Tumors derived from PC3-neo and PC3-bcl-2 cells grew at equal rates when treated with TNP-470, suggesting that this angiogenesis-inhibiting drug eliminated the growth advantage bcl-2 overexpression usually confers on tumors. Together, these data support the hypothesis that increased VEGF production and the increased vascularization associated with this increased expression are essential components of the in vivo growth advantage conferred to tumors by overexpression of bcl-2.
bcl-2 overexpression is associated with the development of androgen-independent prostate carcinomas as well as with increased resistance of such tumors to chemotherapy (12,27,28). One new therapeutic approach that has been proposed to treat prostate tumors is to inhibit their expression of bcl-2, which would restore the sensitivity of such tumors to apoptosis (29,30). Our results indicate that such an approach might have a dual effect on prostate tumors, by inhibiting angiogenesis as well as resensitizing them to apoptosis. In addition, our data suggest that therapies that inhibit angiogenesis may be effective tools in the treatment of prostate cancer.
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NOTES |
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Manuscript received April 19, 2000; revised November 14, 2000; accepted November 27, 2000.
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