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[Cancer Research 59, 1417-1421, April 1, 1999]
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[Cancer Research 59, 1417-1421, April 1, 1999]
© 1999 American Association for Cancer Research

Advances in Brief

Superiority of Yeast over Bacterial Cytosine Deaminase for Enzyme/Prodrug Gene Therapy in Colon Cancer Xenografts1

Els Kievit, Eric Bershad, Emily Ng, Phiroze Sethna, Inderjeet Dev, Theodore S. Lawrence and Alnawaz Rehemtulla2

Department of Radiation Oncology, Division of Radiation and Cancer Biology, University of Michigan Medical Center, Ann Arbor, Michigan 48109 [E. K., E. B., E. N., T. S. L., A. R.], and Glaxo Wellcome, Durham, North Carolina 27709 [P. S., I. D.]


   ABSTRACT
Top
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 
The enzyme/prodrug strategy using bacterial cytosine deaminase (bCD) and 5-fluorocytosine (5-FC) is currently under investigation for cancer gene therapy. A major limitation for the use of bCD is that it is inefficient in the conversion of 5-FC into 5-fluorouracil. In the present study, we show that the Km of yeast cytosine deaminase (yCD) for 5-FC was 22-fold lower when compared with that of bCD. HT29 human colon cancer cells transduced with yCD (HT29/yCD) were significantly more sensitive to 5-FC in vitro than HT29 cells transduced with bCD (HT29/bCD). In tumor-bearing nude mice, complete tumor regression was observed in 6 of 13 HT29/yCD tumors in response to 5-FC treatment (500 mg/kg i.p. daily, 5 days a week for 2 weeks), whereas 0 of 10 HT29/bCD tumors were cured. Our study demonstrates an improved efficacy of the CD/5-FC treatment strategy when yCD was used. This enzyme has, therefore, a high potential to increase the therapeutic outcome of the enzyme/prodrug strategy in cancer patients.


   Introduction
Top
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 REFERENCES
 
5-FU3 is one of the most active chemotherapeutic agents known for the treatment of colorectal cancer. However, the efficacy of systemic treatment with 5-FU is limited by gastrointestinal and hematological toxicity (1) . One strategy to circumvent this systemic toxicity involves the development of enzyme-prodrug gene therapy using the nonmammalian enzyme CD and 5-FC. When the CD gene is introduced into tumor cells, the local conversion of the nontoxic agent 5-FC into the toxic drug 5-FU should reduce the toxicity to normal distal tissues.

In most reported studies using the CD/5-FC treatment strategy, bacterially derived CD has been used. Although some promising results have been obtained in various tumor models using CD-transduced cell lines (2 , 3) or an adenovirus-mediated delivery of CD (4 , 5) , other studies have demonstrated a poor therapeutic effect (6, 7, 8) . The limited success of bacterial CD for gene therapy can partly be explained by the low efficiency of bacterial CD to convert 5-FC into 5-FU, because this is 20-fold less when compared with that of its natural substrate cytosine (9) .

CDs have been found in bacteria and in fungi. Previous studies have reported that CD obtained from yeast is efficient in converting 5-FC into 5-FU (10) . As 5-FC is clinically a useful antifungal agent but not an antibacterial drug, we hypothesized that CD obtained from yeast would more efficiently convert 5-FC into 5-FU, resulting in a higher cytotoxicity to tumor cells. To test this hypothesis, we have isolated and characterized bCD and yCD. The efficacy of both enzymes in the enzyme-prodrug strategy was then compared in vitro and in vivo using established CD-expressing HT29 human colon cancer cell lines. Our results demonstrate that the use of yCD significantly increases the therapeutic effect of the CD/5-FC treatment strategy.


   Materials and Methods
Top
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 REFERENCES
 
Vectors and Cell Lines.
The entire coding sequence of bCD including an extra Kozak sequence followed by an ATG codon was isolated by PCR (11) . The yCD encoding sequence (12) with modifications to facilitate expression in mammalian cells was kindly provided by Glaxo Wellcome (Durham, NC) and amplified by PCR. For both bCD and yCD, unique EcoRI and SalI restriction sites were added to the 3' and 5' ends, respectively.

The human colon cancer cell line HT29 was grown in RPMI supplemented with 10% heat-inactivated calf serum, 2 mML-glutamine, 100 IU/ml penicillin and 100 mg/ml streptomycin. Stable HT29 cell lines expressing either bCD or yCD were generated by viral infection using the retroviral vector LZR. Cells were reseeded 24 h after infection to allow the formation of single colonies, which were subsequently isolated. Stable transduced cell lines were subsequently tested for CD activity. Five HT29/bCD cell lines were obtained, all of which showed similar levels of CD activity. Two HT29/yCD cell lines were isolated, one of which had a slightly higher CD activity and was, therefore, selected for additional experiments. Both the HT29/yCD cell lines were able to convert 5-FC to 5-FU much more efficiently compared with any of the HT29/bCD cell lines.

Enzyme Purification.
bCD and yCD were purified using the GST fusion protein system (13) . The bCD and yCD genes were cloned into the fusion-protein expression vector PGEX-5X-1 directly after the GST gene. To isolate the GST-CD fusion protein the supernatant of lysed transformed Escherichia coli BL21 cells was incubated with glutathione-Sepharose 4B beads as recommended by the manufacturer (Pharmacia Biotech, Piscataway, NJ). Subsequently, Factor Xa (Pharmacia) was added to the beads to cleave off the CD. The supernatant was then incubated with benzamidine Sepharose beads (Sigma, St. Louis, MO) to remove Factor Xa.

Enzyme Activity.
CD activity was quantified by the percentage of conversion of [3H]cytosine or [3H]5-FC (14) . Cell extracts were made by 5 freeze-thaw cycles in 100 mM Tris (pH = 7.8) and 1 mM EDTA. Tumor homogenates were made in 1 ml of medium using a polytron. For the activity assay, 0.2 µg of enzyme, 30 µg of cell extract, or 100 µg of tumor homogenate were incubated with 0.5 mM [6-3H]cytosine or [6-3H]5-FC (1 µCi/mmol, Moravek Biochemicals, Brea, CA) in a 30-µl reaction volume for 2 h at 37°C. The produced [3H]uracil and [3H]5-FU were isolated by elution from a SCX Bond Elute column (Varian, Harbor City, CA) and counted. Also, 0.5 mM [6-3H]cytosine and [6-3H]5-FC were counted and defined as total count (TC). The percentage conversion was calculated as:

The maximum percentage conversion that could be measured with this assay was 80–85%.

For the determination of the Km and Vmax, bCD and yCD were incubated at 37°C with various concentrations of cytosine (Sigma) or 5-FC (Sigma). Samples were taken at various time points, quenched in 1 N HCL and measured spectrophotometrically (15) . For the calculation of the Km and Vmax, the results were plotted as the produced concentration of uracil or 5-FU in 10 min versus the substrate concentration, and were fitted using GraphPad Prism software.

Generation of Antibodies against bCD and yCD.
Polyclonal antibodies against bCD and yCD were generated in rabbits by Berkeley Antibody Company (BabCO, Richmond, CA). Rabbits were immunized with 500 µg of pure bCD or yCD in Freund’s complete adjuvant and boosted with 250 µg in Freund’s incomplete adjuvant every 3 weeks until a satisfactory antibody titer was measured in serum by ELISA. The final titer of the anti-bCD antibody was 1.64 x 104 and for the anti-yCD antibody >1 x 106.

Western Analysis.
Pure enzymes (25–50 ng), cell extracts (15 µg), or tumor homogenates (15 µg) were resolved on a 15% SDS polyacrylamide gel. After blotting, the membrane was blocked with 5% milk in tris-buffered saline containing 0.1% Tween, followed by a 2- incubation with the rabbit anti-bCD serum (1/5000) or rabbit anti-yCD serum (1/106). The secondary horseradish peroxidase-labeled goat antirabbit IgG (1/2500) was added for 1 h. The CD protein was visualized using the supersignal chemiluminescent substrate of Pierce (Rockford, IL).

5-FC and 5-FU Cytotoxicity in Vitro.
The sensitivity of HT29, HT29/bCD and HT29/yCD cells to 5-FC and 5-FU was determined using a standard clonogenic assay (16) . Cells were treated with 5-FC or 5-FU at various concentrations for 24 h at 37°C in conditioned media with 10% dialyzed serum. The surviving fraction was plotted against the concentration of 5-FC or 5-FU and was fitted using the linear-quadratic equation.

Animal Model.
Nude female mice (Nu/Nu CD-1, Charles River Laboratories, Wilmington, MA) of 7–8 weeks received injections s.c. in the flank with 5 x 106 viable tumor cells. Tumors were measured biweekly with calipers in 2 dimensions. Tumor volumes were calculated in mm3 using the formula: (3.14/6) (L x W2). When tumors were >50 mm3 and measured an average volume of 100–150 mm3, treatment was started. Mice received injections daily i.p. with 500 mg/kg 5-FC or 25 mg/kg 5-FU 5 days a week for 2 weeks. Differences in the efficacy between treatments were expressed as the SGD and as the number of complete tumor regressions. For the determination of the SGD, two tumor-volume doubling times (Td) were used as the end point. The SGD was calculated according to the formula:

Complete regression (CR) of tumors was defined as tumors that did not recur for 60 days.

Statistics.
Means (±SE) of at least 3 experiments are expressed. Tumor regressions between HT29/bCD and HT29/yCD tumors were compared using Fisher’s exact test for proportions.


   Results
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 REFERENCES
 
Purification and Characterization of bCD and yCD.
SDS gel electrophoresis of the purified enzymes showed a Mr of 48,000 for bCD and 17,000 for yCD (Fig. 1A)Citation . Native gel analysis of the enzymes revealed that bCD was a pentamer and yCD a trimer. Kinetic studies showed that the velocity of the conversion of the substrate was proportional to the amount of enzyme used. For the determination of the Km and the Vmax, 1 µg/ml and 10 µg/ml of bCD was used for cytosine and 5-FC, respectively. yCD was used at 0.5 µg/ml for both substrates (Fig. 1B)Citation . Although the Kms for cytosine were comparable, the Km of bCD for 5-FC was 22-fold higher than that of yCD (Table 1)Citation . Correspondingly, the Vmax of yCD for cytosine and 5-FC was 3.5- to 6-fold higher than that of bCD. The conversion efficiencies of both enzymes for cytosine and 5-FC were consistent with their Km values. Under standard experimental conditions, both enzymes were equally active in the conversion of cytosine but yCD was much more efficient in the conversion of 0.5 mM 5-FC (Table 1)Citation .



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  		Fig. 1. A, SDS gel electrophoresis of GST-bCD (2 µg, Lane 1), pure bCD (2 µg, Lane 2); GST-yCD (2 µg, Lane 3); pure yCD (5 µg, Lane 4) and GST (2 µg, Lane 5). Samples were resolved on a 15% separation gel under reducing conditions and stained with Coomassi Brilliant Blue. A molecular weight marker (kD) is shown on the left. B, the efficiency of pure bCD ({square}, {blacksquare}) and yCD ({circ}, •) to convert cytosine (open symbols) and 5-FC (closed symbols). The percentage of produced uracil or 5-FU was measured spectrophotometrically for up to 1 h. With respect to the cytosine conversion, 1 µg/ml of bCD or 0.5 µg/ml of yCD was incubated with 1 mM cytosine. To analyze the 5-FC conversion, 10 µg/ml of bCD was incubated with 10 mM, and 0.5 µg/ml of yCD was incubated with 1 mM 5-FC. C, thermostability of pure bCD and yCD. Enzymes were stored at 37°C for up to 168 h, and the enzyme activity was determined at various time points. The activity is expressed as the percentage conversion of substrate by 0.2 µg enzyme in 2 h. {square} and {blacksquare} represent the activity of bCD and {circ} and • represent the activity of yCD using cytosine (open symbols) or 5-FC (closed symbols) as substrate. The average (±SE) of 5 experiments is shown.

 

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  		Table 1 Characteristics of purified bCD and yCDa

 
As yCD has been reported to be unstable at higher temperatures, the thermostability of bCD and yCD was studied. The enzymes were stored at 37°C and the residual enzyme activity was measured at various time intervals (Fig. 1C)Citation . bCD retained 100% of its activity after storage at 37°C for 168 h. In contrast, the activity of yCD decreased by 30–40% after 8 h of storage and was within background levels after 96 h. Both enzymes were stable for at least 3 months when stored at 4°C.

Generation and Characterization of HT29/bCD and HT29/yCD Cell Lines.
To verify that yCD was also more efficient in the conversion of 5-FC than bCD when expressed in mammalian cells, CD-expressing HT29 cell lines were made by retroviral infection. A Western blot analysis of the cell extracts demonstrated that HT29/bCD cells contained a Mr 48,000 anti-bCD immunoreactive band, whereas HT29/yCD cells contained a Mr 17,000 anti-yCD immunoreactive band (Fig. 2A)Citation . These molecular weights were in correspondence with those of the bacterially derived purified enzymes.



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  		Fig. 2. A, Western analysis of pure enzymes and extracts of HT29, HT29/bCD, and HT29/yCD cells. Enzymes (25–50 ng) and cell extracts (30 µg) were resolved on a 15% SDS-PAGE gel, transferred to an immobilon membrane, and incubated with rabbit anti-bCD (left) or rabbit anti-yCD (right) polyclonal antibody. Molecular weight markers (in thousands) are shown on the left. Arrows, the presence of bCD (Mr 48,000) and yCD (Mr 17,000). B, CD activity of HT29/bCD and HT29/yCD cells using cytosine (open bars) or 5-FC (closed bars) as substrate. The activity is expressed as the percentage conversion of substrate by 30 µg of cell extract in 2 h. Average values (±SE) of 16 cell extracts are shown for each cell line. C, cytotoxicity of 5-FC in cultured HT29/bCD ({blacksquare}) and HT29/yCD (•) cells. Cells were exposed to various concentrations of 5-FC for 24 h and assessed for clonogenicity. The average values of 3–6 experiments (±SE) are shown. D, cytotoxicity of 5-FU in cultured HT29/bCD ({blacksquare}) and HT29/yCD (•) cells. Cells were exposed to various concentrations of 5-FU for 24 h and assessed for clonogenicity. The average values of 3–6 experiments (±SE) are shown.

 
Although we made an effort to select clones that expressed similar levels of bCD and yCD, HT29/bCD cells were more effective in the conversion of cytosine than HT29/yCD cells. This difference in conversion efficiency was most likely due to a higher level of expression of the bCD transgene, because the amount of cell extract that was necessary to convert 50% of cytosine in 2 h was approximately 3-fold lower for HT29/bCD cells when compared with HT29/yCD cells. However, despite the higher expression of bCD, HT29/bCD cells were about 10-fold less effective in the conversion of 5-FC than HT29/yCD cells (Fig. 2B)Citation .

5-FC and 5-FU Cytotoxicity in Vitro.
We next examined whether the ability of yCD to convert 5-FC more efficiently than bCD also resulted in an enhanced sensitivity of HT29/yCD cells to 5-FC when compared with HT29/bCD cells. Cells were treated with various concentrations of 5-FC and the cytotoxicity was defined by determining the fraction of surviving cells. HT29/yCD cells were indeed more sensitive to 5-FC than HT29/bCD cells (Fig. 2C)Citation . During a 24-h incubation period, approximately 221 ± 19 µM of 5-FC caused a 50% reduction in the surviving fraction of HT29/bCD cells, whereas only 59 ± 7 µM 5-FC was required to kill 50% of HT29/yCD cells.

To determine whether the difference in cytotoxicity of 5-FC was due to differences in the sensitivity of the cell lines to 5-FU, we studied the cytotoxicity of 5-FU for both cell lines. It appeared that HT29/bCD cells were actually more sensitive to 5-FU than HT29/yCD cells (Fig. 2D)Citation . The concentration of 5-FU that caused a 50% reduction in cell survival was 7.7 ± 1 µM for HT29/bCD cells and 20 ± 2 µM for HT29/yCD cells.

5-FC Cytotoxicity in Vivo.
Although the in vitro data demonstrated a superiority of yCD over bCD for use in the CD/5-FC treatment strategy, these results had to be confirmed in vivo. Mice bearing established HT29/bCD or HT29/yCD tumors received injections daily 5 days a week for 2 weeks with 500 mg/kg 5-FC. Only a small tumor growth delay was observed in HT29/bCD tumors (Fig. 3)Citation . HT29/yCD tumors were significantly more sensitive to 5-FC as is shown by the observed tumor regression. More importantly, no complete regressions were seen in HT29/bCD tumors, whereas tumor cures were observed in 6 of 13 HT29/yCD tumors (P < 0.05).



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  		Fig. 3. Tumor growth after 5-FC treatment in mice bearing HT29/bCD (A) or HT29/yCD (B) xenografts. Mice were treated i.p. daily with 500 mg/kg 5-FC at days 0–4 and 7–11. The average volumes (±SE) relative to those at day 0 of control tumors (open symbols, n = 11–17) and treated tumors (closed symbols, n = 11–13) are shown. CR, complete responses to 5-FC treatment.

 
To determine whether factors other than the differing efficiency of the enzymes to convert 5-FC into 5-FU could explain the difference in response to 5-FC-treatment in vivo, we studied the expression and activity of the bCD and yCD in tumors, as well as the sensitivity of HT29/bCD and HT29/yCD tumors to 5-FU treatment in vivo. Western analysis showed that both enzymes were expressed in the tumors. The conversion efficiency of HT29/bCD tumors for cytosine and 5-FC was 51.9 ± 4.5% and 5 ± 0.3%, respectively, whereas the conversion efficiency for HT29/yCD tumors was 27.6 ± 1.6% and 38 ± 2%. When mice bearing HT29/bCD or HT29/yCD xenografts were treated with 5-FU 5 days a week for 2 weeks at the maximum tolerated dose of 25 mg/kg (2) , only a small tumor growth delay was observed in both of the xenografts and none of the tumors regressed. A slightly longer growth delay was observed in HT29/bCD tumors (SGD = 1.0, n = 18) when compared with HT29/yCD tumors (SGD = 0.4, n = 20). This was in correspondence with the higher sensitivity of HT29/bCD cells to 5-FU observed in vitro.


   Discussion
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
REFERENCES
 
In an effort to improve the efficacy of the CD/5-FC strategy for cancer gene therapy, we have described the characterization and efficacy of a yCD. yCD bears little homology with the previously described bacterial enzyme (bCD) at the DNA sequence level (12 , 17) and at the protein level (bCD is Mr 48,000 and yCD is Mr 17,000). Because yCD is efficient at deaminating 5-FC to yield 5-FU, whereas bCD has a poor conversion efficiency (9 , 10) , we have purified both enzymes to compare their efficacy in the enzyme/prodrug treatment strategy. Data presented here show that yCD and bCD have a comparable Km and Vmax for cytosine, but yCD has a 22-fold lower Km and a 4-fold higher Vmax for 5-FC than bCD. Furthermore, in vitro and in vivo data clearly demonstrated an improvement of the therapeutic effect of the CD/5-FC treatment strategy in CD-transduced HT29 tumor cells when yCD was expressed instead of bCD. This was observed despite the fact that the HT29/bCD clone that we selected expressed more CD protein than the HT29/yCD clone.

Although the enzymatic properties differ between cytosine deaminases obtained from various organisms, the molecular weights, Km and Vmax values, and stabilities of bCD and yCD that were found in our study are in correspondence with the reported characteristics of bCD and yCD that were obtained from their natural sources (10 , 15 , 18) . The thermolability of yCD has been a major reason why most reported studies about the CD/5-FC treatment strategy used bCD (10) . However, our studies showed that despite its thermolability, the use of yCD highly improved the therapeutic effect. Indeed, if yCD entered the circulation, it would not be expected to remain stable for a sufficient time to convert 5-FC into 5-FU systemically. This would prevent a possible systemic toxicity, which might occur when the thermostable bCD enters the circulation.

The efficacy of the CD/5-FC treatment strategy in vivo that uses cell lines that are transduced with bCD has been reported by several groups with varying results. One of the first promising studies used CD-expressing WiDr cells (2 , 19) . All of the tumors regressed after treatment with 500 mg/kg 5-FC for 10 consecutive days, but a relapse was observed in 70% of the tumors. These investigators also demonstrated that only 4% of the tumor cells had to be transduced with the CD gene to observe tumor regressions (3) . In contrast, other studies reported only a small tumor growth delay in CD-expressing fibrosarcomas, adenosarcomas, and gliosarcomas when mice were treated with 5-FC (500 mg/kg daily or 37.5 mg per animal twice a day for 10–14 days; Refs. 6 , 7 ). These results are comparable to the observed tumor growth delay in HT29/bCD tumors in our study. The regression of WiDr/bCD tumors in response to 5-FC treatment (2) may be explained by a possible higher expression of bCD, which results in an increased sensitivity to 5-FC. The IC50 for 5-FC of WiDr/bCD cells was indeed approximately 8-fold lower than that of our HT29/bCD cells (27 µMversus 221 µM).

Although the use of tumors consisting of stable CD-transduced tumor cells is a valid model to study the effect of the conversion efficiency of CD on the therapeutic outcome of the CD/5-FC treatment strategy, this model may not reflect the clinical situation inasmuch as current gene-delivery systems have a poor transduction efficiency. Because only 5–10% of the tumor cells are infected using adenovirus, a potent bystander effect of the CD/5-FC treatment strategy is required. Previously, we have demonstrated that bCD-expressing tumor cells were killed more effectively by 5-FC than bystander nontransduced cells because of the high intracellular concentration of 5-FU, which could result in a decreased bystander effect (11) . Because yCD is more efficient than bCD in the conversion of 5-FC, the preferential killing of transduced cells may be more prominent, resulting in a reduction of the bystander effect to a higher extent. However, our in vitro data have demonstrated an improved bystander effect of HT29/yCD cells in response to 5-FC treatment when compared with HT29/bCD cells.4 Although a preferential killing of HT29/yCD cells was observed, the higher efficiency of yCD to convert 5-FC had most likely resulted in the production of a sufficient amount of 5-FU to kill the bystander cells.

In conclusion, the present study demonstrates that the use of yCD significantly improves the efficacy of the CD/5-FC treatment strategy, and that complete tumor regressions were observed in 60% of the tumors. Although the efficacy of 5-FC and of 5-FU treatments was only slightly different in HT29/bCD tumors, the major difference in their efficacy in HT29/yCD tumors validates the advantage of the preferential use of yCD in the enzyme/prodrug strategy as well as the use of the CD/5-FC treatment strategy over conventional chemotherapy with 5-FU. Because 5-FU is not only a chemotherapeutic agent but also a radiosensitizer (20) , the combination of the CD/5-FC treatment strategy and radiotherapy has a high potential to improve the therapeutic outcome in cancer patients (21, 22, 23) . We are, therefore, currently comparing bCD and yCD in this combination treatment strategy.


   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 study is supported by NIH Grant CA80145 and a Munn Research Award UMCCC. Back

2 To whom requests for reprints should be addressed, at Department of Radiation Oncology, University of Michigan Medical Center, 1331 E. Ann Street, Ann Arbor, MI 48109-0582; E-mail: alnawaz{at}umich.edu Back

3 The abbreviations used are: 5-FU, 5-fluorouracil; 5-FC, 5-fluorocytosine; CD, cytosine deaminase; bCD, bacterial CD; yCD, yeast CD, yeast-derived CD; GST, glutathione-S-transferase; SGD, specific growth delay. Back

4 A. Rehemtulla, E. Kievit, M. A. Davis, E. Ng, and T. S. Lawrence, Extracellular expression of cytosine deaminase results in increased 5-FU production from 5-FC treatment, manuscript in preparation. Back

Received 12/ 3/98. Accepted 2/11/99.


   REFERENCES
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 

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