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Departments of Orthopedic Surgery [M. R., W. P., M. G., C. L., D. R. C.], Preventive Sciences [P. M.], and Pediatrics [P. O.], University of Minnesota, Minneapolis, Minnesota 55455, and Department of Pediatric Oncology, Alberta Children’s Hospital, Calgary, Alberta, T2T 5C7 Canada [V. L.]
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The CD enzyme/prodrug system has advantages over other enzyme/prodrug systems. One advantage is that 5FU can function as a cytotoxic molecule and as a radiosensitizer (5) . This makes the CD system particularly attractive for treating tumors that will receive radiation treatment such as colon cancer. It has been shown in experimental models that CD-based prodrug systems are effective in killing colon cancer cells (2 , 3 , 6 , 7) , and recently, a synergistic effect has been reported with combined 5FC treatment and radiation (4 , 8) . A second advantage of the CD enzyme prodrug system is 5FU-directed bystander killing (2 , 4 , 6) . Bystander killing occurs when tumor cells not expressing CD are eliminated by passive diffusion of 5FU from adjacent CD-expressing, 5FU-producing cells. An advantage of the bystander effect is making it unnecessary to deliver the gene in all cells to obtain an effect in vivo.
Novel means for reducing the size of extremity sarcomas are needed to improve local treatment of these devastating cancers and to decrease the frequency of limb amputation. In this investigation, a novel fusion gene containing a truncated form of the human NGFR and either the bacterial or yeast CD gene was used to determine whether CD-expressing soft tissue or bone sarcoma cells would respond to 5FC treatment. Both direct and bystander tumor killing were evaluated. In addition, as osteolysis plays a pathological role in bone sarcoma progression, we examined the possibility that 5FC treatment of CD-transduced bone sarcomas decreased tumor-induced osteolysis.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Vector Design.
The retrovirus vectors containing the fusion genes comprised of the truncated human NGFR gene and either the bacterial (E. coli) CD gene or the yeast (S. cerevisiae) CD gene, designated NGFR-bCD and NGFR-yCD, respectively, were provided by Dr. Paul Orchard (9)
. Briefly, the NGFR-bCD and NGFR-yCD were constructed using the PCR, eliminating the termination codon of the NGFR gene and incorporating a [gly4ser]2 linker before the CD genes that had the initial ATG sequence eliminated. The modified CD genes were subcloned into a pCR2.1 vector (Invitrogen Life Technologies, Inc., Carlsbad, CA) and sequenced to confirm its identity. The fusion genes were excised from pCR2.1 and subcloned into the LXSN vector (10)
. Clones were screened to confirm the correct orientation. The final constructs, termed LNG/bCDSN and LNGyCDSN, were transfected into the PA317 packaging line, and 0.4 mg/ml G418 (Life Technologies, Inc.) were used to select positive clones (11)
. High-titer clones were chosen to generate viral supernatants, which were used to transduce the 2472 tumor line.
Retrovirus Transduction.
Tumor cells were plated at 105 cells in a 100-mm dish and were transduced with a 2-ml aliquot of viral supernatant containing 20 µg/ml DEAE-Dextran. Cells were cultured for 4 h with rocking before 6 ml media was added. Cells were grown for 48 h before changing to selection media, which contained 0.8 mg/ml G418. Transduced 2472 tumor cells were designated 2NGFR-bCD and 2NGFR-yCD, with 2 indicating 2472 cells. Transduced tumor cells were maintained as the parent lines, except for the addition of 0.8 mg/ml G418.
FACS Analysis.
Transduced tumor cells were harvested at passage, and an aliquot of 106 cells was used for FACS analysis. Briefly, the cells were pelleted in 12 x 75-mm tubes, rinsed once in PBS containing 0.1% BSA, and divided into two 100-µl tubes containing either 100 ng/106 cells of a biotinylated monoclonal mouse antihuman NGFR (clone 20.4, provided Dr. P. Orchard) or an equal amount of a biotinylated isotype control immunoglobulin. Cells were incubated on ice for 30 min, washed once in 10-fold volume of dPBS containing 0.1% BSA, followed by addition of 100 ng/106 cells of the secondary antibody conjugated to Streptavadin-PE (BD Biosciences PharMingen, San Diego, CA). Cells were returned for an additional 30 min of incubation on ice, followed by a third wash and resuspension in 0.5 ml of 4% paraformaldehyde in dPBS. Analysis of the presence of the NGFR antigen on cells was performed using a FACScaliber (BD Biosciences Immunocytometry Systems, San Jose, CA) flow cytometer for acquisition and FLOJO software for quantitation, plotting the isotype control or nontransduced cell line against NGFR-expressing cells. For positive sorting, 20 x 106 cells were pelleted into a sterile 12 x 75-mm tube and labeled as above using 2 µg of antibody in a total volume of 1 ml. The brightest 20% NGFR-positive cells were recovered using a FACSVantage flow cytometer. Positive cells were subcultured, maintained as heterogeneous lines, and designated 2NGFR-yCD+.
Western Analysis.
Cells from transduced and nontransduced cell lines (at least 107 total) were treated with trypsin, pelleted, and washed with PBS. Cell pellets were resuspended in 0.5 ml ice-cold lysis buffer [50 mM Tris-HCl (pH 7.6), 150 mM NaCl, 5 mM EDTA, 0.5% Triton X-100, 0.2% SDS, 1 mM NaF, and 1 mM Na3PO4] with protease inhibitor mixture tablets (Roche, Indianapolis, IN), then incubated on ice for 20 min and centrifuged 2 min at full speed in a microcentrifuge at 4°C. Supernatant was stored at -80°C. Two hundred µg of cell lysate/lane were separated on a 10% reducing SDS-PAGE and transferred to a nitrocellulose membrane (Schleicher and Schuell, Keene, NH). The membranes were blocked with 5% nonfat milk in PBS containing 0.05% Tween 20 (PBST) for 1 h at room temperature and incubated with primary antibodies overnight at 4°C. Polyclonal antihuman NGFR (p75) antibody (R&D Systems, Inc., Minneapolis, MN) and polyclonal anti-CD (Bio-Trend, Destin, FL) were applied at dilutions of 1:500 and 1:1000, respectively. Membranes were washed three times for 10 min in PBST before 1-h room temperature incubation with HRPs and conjugated secondary antibodies (bovine antigoat-HRP, 1:2000 or bovine antisheep-HRP, 1:3000; Santa Cruz Biotechnology, Santa Cruz, CA). After six 5-min washes in PBST and one 20-min wash in PBS, the membranes were incubated with enhanced chemiluminescence (Pierce, Rockford, IL) for 1 min before exposure to Kodak X-Omit AR film. Membranes were reprobed with antitubulin as a loading control.
In Vitro Cytotoxicity Assay.
Cell counts and viability were assessed using a tetrazolium [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] dye conversion assay (Promega, Madison, WI). To assess drug cytotoxicity, 5 replicate wells containing 2.5 x 103 cells in 0.2 ml were cultured in 96-well plates with various dilutions of 5FU (1–20 µM) or 5FC (0.025–10.0 mM). Cells were demi-depleted at 3 days, a second dose of drug was added and cells returned to incubation for an additional 3 days. Dye was added on day 6 and allowed to react for 2 h at 37°C. Dye conversion was read using an ELISA plate reader at 490 nm against 610 nm. Cytotoxicity was determined by calculation of the number of viable cells as measured against wells containing no drug. Drug cytotoxicity experiments were performed on each transduced and nontransduced cell line in at least three distinct experiments, and average data were used to plot in vitro killing. Bystander killing was assessed, as above, using various ratios of parental and transduced cells.
In Vivo Tumor Killing.
A total of 12 C3H/HeJ (National Cancer Institute) or NOD.CB17-PrkdcSCID (Jackson Labs, Bar Harbor, ME) mice were used for each tumor or treatment group. The Animal Care and Use Committee of the University of Minnesota approved all experimental protocols. Just before implantation, tumor cells were trypsinized, pelleted, rinsed in dPBS, and resuspended at appropriate concentrations in dPBS containing 1% BSA. Tumor cells were implanted either s.c. or in the left femur intramedullary space.
s.c. injection of 5 x 106 tumor cells was performed over the left flank in a total volume of 200 µl. To facilitate tumor measurements, a small patch of fur was shaved over the implant site with tumor length and width measured every other day starting at day 6. Tumor volume was calculated using the formula for the volume of an ovoid: volume = 4 ÷ 3 x p x (length ÷ 2) x (width ÷ 2) (2)
. Treatment with 5FC, 400 mg/kg/day (dissolved as 12.5 mg/ml in 0.9% sterile NaCl, injection grade), or 5FU (10, 25, or 50 mg/kg, diluted in 0.9% saline) was started when tumors reached 
100 mm3. Mice were euthanized 11 days later. At tissue harvest, mice and tumors were weighed, tumors were measured, and s.c. tumors fixed in Z-fix (Anatech, Battle Creek, MI) for histology. Bystander effect was evaluated in s.c. tumors in C3H/HeJ mice, as described above, with a mixture of 90% (4.5 x 106) 2472 cells and 10% (0.5 x 106) nonsorted or sorted 2NGFR-yCD cells.
Intraosseous injections of tumor cells were performed as described previously (12) . In brief, injection of 105 2NGFR-yCD cells in a 20-µl aliquot of dPBS containing 1% BSA was performed through a knee arthrotomy on mice anesthetized with a ketamine/xylazine mixture (100 and 7.5 mg/kg, respectively). Treatment with 5FC commenced 7 days after tumor cell injection, and the animals were sacrificed 9 days later. Radiographs (Faxitron MX-20 cabinet X-ray machine and Kodak Min-R 2000 film) of femora (x5) were performed and bone destruction scores were assessed as follows: 0, no bone loss; 1, intramedullary osteolysis without full thickness cortical osteolysis; 2, intramedullary osteolysis and full thickness cortical erosion through one cortex; and 3, intramedullary osteolysis and full thickness cortical erosion through both cortices (13) . Femora were fixed in Z-fix for 24 h, decalcified in neutral 10% EDTA for 10 days, and processed for histology.
Histology, Histomorphometry, and Immunohistochemical Analysis.
Tumors and decalcified femora were embedded in paraffin and cut at 5 µm. Routine H&E stain was performed on all specimens, and four replicate sections were used for histomorphometric measurements. Images of whole femora were acquired using a Spot 2 digital charge-coupled device (CCD) on a Nikon E-1000 microscope at x1 and ImagePro Plus (Media Cybernetics Inc., Silver Spring, MD) used to quantitate the area of bone containing tumor.
Immunohistochemistry for NGFR was performed on deparaffinized and hydrated sections of s.c. tumor and femora. Staining was a standard three-step avidin-biotin complex procedure. Briefly, monoclonal mouse antihuman NGFR (clone 20.4, provided by Dr. P. Orchard) was diluted 1:2000 and incubated at 4°C overnight. A biotinylated goat antimouse IgG F(ab')2 (Rockland, Gilbertsville, PA) was used as the secondary antibody (1:1000 dilution), and the tertiary reagent was HRP avidin-biotin complex (HRP-avidin-biotin complex method; Vector Labs, Burlingame, CA). 3,3'-Diaminobenzidine (Zymed, San Francisco, CA) was used to develop the staining reactions, and sections were lightly counterstained with Mayer’s hematoxylin, dehydrated, and coverslipped using Permount. Epitope retrieval involved 15-min rinses with first, 0.05% saponin, and second, 0.1% Triton X-100. All antibody incubations were followed by three rinses with dPBS. A nonspecific antibody (dPBS containing 10% horse sera), and avidin/biotin blocking steps were performed before addition of the primary antibody. Nonspecific peroxidase activity was blocked by incubation with 3% hydrogen peroxide (10 min) after the secondary antibody.
Statistics.
In vitro data are presented as means ± SD and in vivo data as means x SE. Statistical significance was determined by Student’s t test. P of <0.05 was considered statistically significant.
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RESULTS |
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60% of the transduced cells expressed surface NGFR (Fig. 2A)
. Comparison of 2NGFR-bCD and 2NGFR-yCD FACS expression profiles showed an approximate 4-fold greater expression in 2NGFR-yCD cells compared with 2NGFR-bCD cells. NGFR protein was detected in each transduced sarcoma and levels of protein expression paralleled FACS data. NGFR-bCD was a single band at apparent molecular weight of Mr 95,000 and NGFR-yCD had two bands with apparent molecular weights of Mr 67,000 and Mr 63,000. NGFR protein levels were higher in 2NGFR-yCD-expressing cells than in 2NGFR-bCD cells, and NGFR protein levels were higher in 2NGFR-yCD cells selected by FACS for NGFR expression (2NGFR-yCD+) compared with cells selected by neomycin alone (Fig. 2B)
. Western analysis detected yCD protein in 2NGFR-yCD cells (Fig. 1D)
and bCD in 2NGFR- bCD cells (Fig. 2C)
. Taken in total, these findings indicate that the NGFR-bCD and NGFR-yCD fusion genes produce intact NGFR-CD proteins of expected size and that cell surface expression of NGFR can be used for positive selection of cells transduced with the NGFR-yCD or NGFR-bCD fusion gene.
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. Comparison of sarcoma cells transduced with NGFR-bCD and 2NGFR-yCD indicated that cells transduced with NGFR-yCD were more sensitive to 5FC treatment. After exposure to 0.75, 1.0, and 1.25 mM 5FC, there were significantly increased numbers of 2NGFR-bCD cells compared with 2NGFR-yCD cells. After treatment with 0.75 mM 5FC, >50% of 2NGFR-bCD cells were alive, compared with 15% of 2NGFR-yCD (Fig. 3B)
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Evaluation of Bystander Killing in Vitro.
2NGFR-yCD cells killed untransduced 2472 cells through a bystander effect. Bystander killing was examined in vitro by culturing parent 2472 cells with 2NGFR-yCD cells in various concentrations and exposing the cells to increasing doses of 5FC. Bystander killing occurred with as few as 10% 2NGFR-yCD cells (Fig. 3C)
. ED50 increased with decreasing percentages of 2NGFR-yCD cells. Cultures containing 100, 50, and 10% 2NGFR-yCD cells had ED50 of 0.01, 0.05, and 0.25 mM 5FC, respectively. Finally, 2.5 mM 5FC eliminated >85% of tumor cells with each mixture of transduced and parent sarcoma cells (Fig. 3C)
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Effect of 5FU on s.c. 2472 Tumors.
5FU treatment affected growth of s.c. sarcomas. Animals with s.c. 2472 tumors were treated with 10, 25, and 50 mg 5FU/day. Although 10 mg/kg/day 5FU had no effect on tumor volume, doses of 25 and 50 mg/kg/day affected tumor growth. Treatment with 25 mg/kg/day halted tumor growth and treatment with 50 mg/kg/day decreased tumor volume compared with initial values. When compared with saline treatment, significant decreases in tumor volume were present 7 days after 5FU treatment (25 and 50 mg/kg/day). At the end of each experiment, there was a >100% reduction in the tumor volume among mice treated with 25 or 50 mg 5FU/day compared with mice receiving no treatment (Fig. 4A)
. Corresponding reduction in tumor weights were observed. Final tumor weights for saline, 10, 25, and 50 mg/kg 5FU/day being 0.6 ± 0.4, 0.6 ± 0.1, 0.08 ± 0.06, and 0.04 ± 0.02, respectively (Table 1)
. When final tumor volumes were compared with tumor volumes before 5FU treatment, only the highest dose of 5FU (50 mg/kg/day) was effective at decreasing the initial tumor volume (Fig. 4A)
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). 5FU treatment also caused animal deaths. Although no animals treated with saline, 10, or 25 mg/kg/day 5FU died, all mice treated with 50 mg/kg/day 5FU died before the experiment’s end (17 days).
Effect of 5FC on s.c. 2NGFR-CD Tumors.
5FC treatment decreased growth of s.c. 2NGFR-yCD tumors. Mice with 2472, 2NGFR-bCD and 2NGFR-yCD tumors were treated with 5FC, and tumor volume and weight were monitored. As expected, tumor volumes in saline-treated mice progressively increased over time. 5FC treatment had no effect on 2NGFR-bCD tumors but showed profound effect on 2NGFR-yCD tumors. 5FC treatment in animals with 2NGFR-yCD tumors reduced tumor volumes below pretreatment size and dramatically decreased tumor volume compared with tumors in saline-treated mice (51 ± 60 versus 938 ± 767 mm3; Fig. 4B
). Corresponding reduction in tumor weights was also seen. Tumor weights from 5FC treated mice were significantly smaller than control (0.6 ± 0.4 versus 0.1 ± 0.09 g, P < 0.001) and were indistinguishable from tumor weights in mice treated with 25 or 50 mg/kg 5FU/day (Table 1)
. In addition, analysis of animal weights and treatment-associated deaths revealed that 5FC treatment had no effect on animal weight and caused no treatment-associated complications (Table 1)
.
To determine whether decreased tumor volume after 5FC treatment represented immunological killing, similar experiments were performed in NOD/SCID mice. Profound reduction in tumor volume was noted when NOD/SCID mice with 2NGFR-yCD tumors were treated with 5FC (n = 9). Eight mice had no detectable tumor, and 1 mouse had a 4-mm3 tumor. In contrast, the mean tumor volume in saline-treated mice was 953 ± 349 mm3. This finding indicates that significant immunological killing was not occurring, and the primary mechanism for tumor killing was a direct cytotoxic effect of prodrug-generated 5FU.
Evaluation of Bystander Killing in s.c. Tumors.
2NGFR-yCD cells killed untransduced 2472 cells in vivo through a bystander effect. The possibility that sarcoma cells transduced with the NGFR-yCD fusion gene could direct bystander killing of nontransduced sarcoma cells was assessed by growing s.c. tumors composed of 10% 2NGFR-yCD cells and 90% 2472 cells and treating tumor-bearing mice with saline or 5FC. As expected, tumor volumes in saline-treated mice progressively increased over time. 5FC treatment slowed tumor growth. After 
7 days of treatment, there was a significant reduction in tumor volumes between saline and 5FC-treated mice (Fig. 4C)
. In addition, after 10 days of treatment, tumor volumes in saline-treated mice were two times greater than tumor volumes in 5FC-treated mice (Fig. 4C)
.
Bystander killing mediated by 2NGFR-yCD cells was significant but incomplete, with residual tumor remaining at the completion of each experiment. We next examined the possibilities that increased NGFR-yCD gene expression would enhance the bystander effect. 2NGFR-yCD cells were sorted by FACS and the highest (20%) NGFR-expressing cells were expanded in culture. They showed a 2-fold increase in the level of 2NGFR-yCD expression as determined by FACS analysis. These sorted cells, designated 2NGFR-yCD+, were mixed 1:9 with 2472 cells and injected as a s.c. tumor inoculum. Treatment of mice with these tumors revealed increased bystander killing (Fig. 4C)
. Specifically, after 12 days of treatment, there was a 5-fold reduction in tumor volume between saline and 5FC-treated mice. However, as with the unsorted 1:9 mix tumors, there was tumor growth despite treatment with 5FC.
We hypothesized that if 5FC treatment eliminated NGFR-yCD-expressing cells and therefore limited the bystander effect. Findings indicated that 5FC treatment of mice with 10% 2NGFR-yCD/90% 2472 sarcomas eliminated NGFR-expressing tumor cells. Immunohistochemical evaluation of tumors that grew from 10% 2NGFR-yCD/90% 2472 cells showed different findings in saline-treated and 5FC-treated mice. As expected, tumors from saline-treated mice had NGFR-expressing cells (Fig. 5B)
. In contrast, NGFR-expressing cells could not be identified in tumors from 5FC-treated mice (Fig. 5A)
. This finding suggests that 5FC treatment was killing 2NGFR-yCD cells before bystander killing could reduce the tumors below pretreatment size.
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. Bone tumor area in 5FC-treated mice was 1.3 ± 4.1 mm2, a significant reduction compared with 11.8 ± 6.0 mm2 in saline-treated mice (P < 0.002).
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Bystander killing of osteoclasts by 2NGFR-yCD bone cancers was profound and prevented cancer-induced bone destruction. Histological sections showed minimal or no bone destruction at sites of osseous tumor injection in mice treated with 5FC. This was a dramatic contrast to effects in bone of saline-treated mice where there was widespread bone destruction (Fig. 6, C and D)
. Radiographic bone destruction scores were assigned to tumor-injected bones from saline- and 5FC-treated mice. All tumor-injected femora from saline-treated mice had radiographic evidence of bone destruction with a mean bone destruction score of 2.8 ± 0.5. In contrast, only 1 of 10 tumor-injected femora from 5FC-treated mice had radiographic evidence of tumor osteolysis. The mean bone destruction score for femora from 5FC-treated mice was 0.2 ± 3 (P < 0.002; Fig. 7
). Histological evaluation of bones showed that femora from saline-treated mice had areas of bone resorption with significant numbers of osteoclasts. Femora from 5FC-treated mice had occasional areas of bone resorption but no osteoclasts, indicating that 5FC treatment had eliminated osteoclasts that previously formed at sites of tumor (Fig. 8)
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DISCUSSION |
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Previous direct comparison of bacterial and yeast CD in animal models report that the yCD systems provide superior tumor killing compared with bCD systems. Kievit et al. (3) studied the effect of bCD and yCD in HT29 human colon cancer cells, reporting that 5FC treatment had no effect on growth of bCD-transduced tumors but caused regression of yCD-transduced tumors. Decreased tumor killing by bCD-transduced HT29 cells was attributed to a 22-fold higher Km for bCD compared with yCD. Circumstances in this study do not permit direct comparison of NGFR-yCD and NGFR-bCD-transduced cells because CD protein was significantly higher in NGFR-yCD-transduced cells.
Our evaluation of treatment-associated morbidity and mortality indicated that the administration of 5FC in association with CD expression had clear advantages over 5FU treatment. This is an important finding because it provides direct evidence supporting the concept that systemic toxicity is minimal, despite the capacity to eliminate CD-expressing enzyme prodrug system has a tumor killing effect. Furthermore, it shows that catastrophic systemic toxicity (death) results with 5FU doses that achieved the equivalent effect as 5FC.
When nontransduced sarcoma cells were mixed with 2NGFR-yCD cells and treated with 5FC, significant killing occurred in vitro and in vivo. 5FC treatment of a mixture of nontransduced and NGFR-yCD cells in vitro dramatically reduced the number of cancer cells. 5FC treatment of s.c. sarcomas grown from a mixture of 2472 and 2NGFR- yCD cells significantly slowed tumor growth. Similarly, in vivo bystander killing and slowed tumor growth has been observed in an experimental s.c. model of HT29 colon cancer, s.c. WiDr colon tumors, and intrahepatic HT29 colon cancers (2 , 8) .
The 2472 sarcoma studied in this article has been shown previously to induce significant bone destruction (osteolysis; Ref. 12 ). The mechanism for this destruction is tumor stimulation of osteoclastic bone resorption caused by stimulation of osteoclast formation and activity. Previous work has defined the benefit of inhibiting tumor-induced osteoclastogenesis and has demonstrated that osteoclast-targeted therapies decrease both cancer-induced bone destruction and cancer-induced pain (15 , 16) . In this investigation, 5FC treatment eliminated osteoclasts at sites of bone sarcomas. This resulted in a dramatic reduction in sarcoma-induced bone destruction and introduces the interesting possibility that tumor-generated 5FU may be exerting bystander killing effects on osteoclasts.
In conclusion, we report that yCD-based enzyme/prodrug therapies can decrease the size of bone and soft tissue sarcomas in an animal model. These findings suggest that the yCD-based therapies may be an effective means for decreasing the size of bone and soft tissue sarcomas, providing the gene can be delivered in vivo. Surgical management of large extremity sarcomas remains challenging and can necessitate limb amputation. Identification of the yCD prodrug system as an experimental means for decreasing sarcoma tumor size provides impetus to explore the possibility that this enzyme prodrug system can be used as an adjuvant therapy for the management of sarcomas. In addition, as the 5FC/CD enzyme/prodrug system has shown efficacy as a radiosensitizer in both 5FU-sensitive and 5FU-resistant tumors (4 , 17 , 18) , it will be important to determine whether the combination of 5FC/yCD enzyme/prodrug system with radiation has a synergistic effect on sarcomas. Obstacles remain, however, and the most formidable of these is identification of an efficient gene delivery system. Concepts to enhance gene delivery that are currently under investigation include local intratumoral injection of nonselective retroviral or adenoviral vectors and viral targeting through engineering of the viral coat (19 , 20) .
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by the National Cancer Institute Grant CA90434 and the National Institute of Arthritis, Musculoskeletal, and Skin Diseases Grants AR43595 and AR47302. The Roby C. Thompson Endowment provided additional financial support. 
2 To whom requests for reprints should be addressed, at University of Minnesota Department of Orthopedic Surgery, 420 Delaware Street, Southeast, MMC 806, Minneapolis, MN 55455. Phone: (612) 626-9934; Fax: (612) 624-0944.
3 The abbreviations used are: 5FC, 5-fluorocytosine; CD, cytosine deaminase; 5FU, 5-fluorouracil; NGFR, nerve growth factor receptor; 2472, NCTC clone 2472 fibrosarcoma; dPBS, Dulbecco’s PBS; FACS, fluorescence-activated cell sorting; HRP, horseradish peroxide.
Received 3/11/03. Revised 7/24/03. Accepted 7/31/03.
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-Tubulin is shown as a loading control.
) and transduced sarcoma cells (2NGFR bCD, 
; 2NGFR yCD, 
) were cultured in the presence of increasing doses of (A) 5FU or (B) 5FC. Combinations of 2472 and 2NGFR yCD cells were cultured with increasing doses of 5FC (C). Data in A and B represent mean ± SD of six separate experiments. Percent designation in (C) represents proportion of 2NGFR yCD cells in 2NGFR yCD:2472 mixture, mean ± SD of five replicates in a single experiment. 
, significantly different (P < 0.01) compared with 2NGFR yCD.
) or increasing doses of 5FU (10 mg/kg/day, ····
····; 25 mg/kg/day, ———
