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[Cancer Research 62, 2337-2342, April 15, 2002]
© 2002 American Association for Cancer Research

Experimental Therapeutics

High and Selective Expression of Yeast Cytosine Deaminase under a Carcinoembryonic Antigen Promoter-Enhancer1

Mukesh K. Nyati, Arun Sreekumar, Shengping Li, Ming Zhang, Susan D. Rynkiewicz, Arul M. Chinnaiyan, Alnawaz Rehemtulla and Theodore S. Lawrence2

Departments of Radiation Oncology [M. K. N., S. L., M. Z., S. D. R., A. R., T. S. L.] and Pathology [A. S., A. M. C.], University of Michigan, Ann Arbor, Michigan 48109-0010


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Yeast cytosine deaminase (yCD)-based gene therapy offers the potential for selective production of the cytotoxic and radiosensitizing drug 5-fluorouracil (5-FU) from the benign prodrug 5-fluorocytosine within colorectal cancers. Although previous attempts to target therapy to colorectal cancer using the carcinoembryonic antigen (CEA) promoter have demonstrated specificity, this has been achieved at the cost of 10- to 300-fold loss in activity compared with strong but nonspecific rous sarcoma virus (RSV) or cytomegalovirus promoters. We developed a highly specific and active gene transfer method for colorectal cancer using CEA under control of a promoter-enhancer. We compared the RSV promoter-derived with the CEA promoter-enhancer-derived transgene expression in 10 different cell lines with differing CEA status. We found that the transgene expression resulting from both transient transfection and adenoviral infection with the CEA promoter-enhancer was as strong as the RSV promoter while maintaining specificity for CEA-producing cell lines. For instance, when we compared yCD expression between LoVo (CEA+) and human fibroblast (CEA-), we found a 30-fold-increased yCD expression in LoVo cells from CEA-enhancer adenovirus although there was no difference in the yCD expression between the cell lines when infected with RSV/yCD virus. This specificity was also achieved while maintaining a higher yCD enzyme activity than we obtained with RSV/yCD adenovirus in an HT-29 intrahepatic tumor model. We then compared the response of HT-29 xenografts to treatment with 5-fluorocytosine and yCD adenovirus driven by either the RSV or the CEA promoter-enhancer and found similar tumor growth inhibition. These findings suggest that the CEA promoter-enhancer strategy confers specificity while preserving activity and is worth exploring in additional animal and, potentially, clinical trials.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
One approach for improving the outcome of treatment for patients with metastatic colorectal cancer involves the use of virus-directed enzyme-prodrug gene therapy (1) . We and others have focused on a gene therapy strategy using 5-FC3 and CD (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15) . CD catalyzes the deamination of the prodrug 5-FC into the cytotoxic and radiosensitizing drug 5-FU. We have shown that, despite having similar activity toward cytosine, yCD is far more efficient at the conversion of 5-FC to 5-FU than is bacterial CD in both in vivo and in vitro models (12) . We have also demonstrated in vivo that a cure rate of >50% can be achieved using 5-FC and radiation if as few as ~10% of tumor cells are expressing yCD (14) .

Although Ad-mediated gene transfer is an efficient method for delivering genetic material into a wide range of cells in vitro and in vivo, a major limitation of this vector in cancer therapy is the nonspecific transduction of therapeutic genes into normal cells (16) . Tumor-specific promoters or regulatory elements such as CEA or AFP have the potential to target suicide genes to gastrointestinal tumors (17) . CEA is normally expressed in fetal tissue and is transcriptionally silent in adults but is frequently overexpressed in human adenocarcinoma (18) . Although the use of CEA to target gene therapy has resulted in specificity, this has come at the cost of a 10- to 300-fold loss in activity compared with nonspecific viral promoters such as CMV or RSV (19, 20, 21, 22, 23) . We hypothesized that it would be possible to maintain the specificity associated with CEA but increase the activity by the use of a promoter-enhancer. To test this hypothesis, we investigated the use of a 2.1-kb fragment derived from the CEA promoter as a means to enhance the basal activity of the CEA promoter. For this purpose, we constructed a recombinant Ad vector carrying the yCD gene under the regulation of a basal CEA promoter (pAdCEAyCD) as well as a CEA promoter with an enhancer sequence placed upstream from the construct pAdCEA-enhancer-yCD. When we found in transient transfection assays that the CEA promoter-enhancer substantially increased yCD activity while maintaining specificity to CEA-expressing cells, we constructed yCD-carrying Ads under the CEA promoter-enhancer and the nonspecific but highly active RSV promoter. Finally, we compared enhanced AdCEA-yCD to AdRSV-yCD in both in vitro and human tumor xenograft studies.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals
(6-[3H]) 5-FC (7.6 Ci/mmol) was purchased from Moravek Biochemicals (Brea, CA), 5-FC from Sigma Chemical Co. (St. Louis, MO), and anti-CEA antibody (Ab-3) from Lab Vision Co. (Fremont, CA).

Cell Culture
Human colon cancer cell lines LoVo, HT-29, NCI-H508, Caco2, a pancreatic cancer cell line BxPC-3, and breast cancer cell line MCF-7, were obtained from American Type Culture Collection. Normal neonatal HFs were isolated in our laboratory; HuH-7 (liver carcinoma) was a generous gift from Dr. Patricia Marion (Stanford University, Stanford, CA). Human UMSCC6 and murine SCCVII cells were obtained from Dr. T. E. Carey at the University of Michigan, Ann Arbor, MI. The LoVo cell line was maintained in Ham’s F-12 medium supplemented with 10% fetal bovine serum (Life Technologies, Inc., Grand Island, NY). The remaining cell lines were grown in RPMI supplemented with 10% bovine serum. The cultures were tested routinely for Mycoplasma infection.

Plasmid Constructions
CEA promoter regions isolated from human genomic DNA, located from -266 and +102 of the transcriptional start site of the CEA gene were used to direct the yCD gene expression in an adenoviral vector pAdMCSloxp. The yCD gene and the Kozak sequence from pZyCD were amplified by PCR using primers 5'-ATG AAG CTT TGT CCA CTC CCA GGT CC-3') and (5'- CTA GAA GGC ACA GTC GAG GTT ACT ACT CAC CAA TAT CTT CAA ACC-3'; Ref. 11 ). The bovine growth hormone (poly)A tail from pSecTag-pA was amplified using primers 5'-GGT TTG AAG ATA TTG GTG AGT AGT AAC CTC GAC TGT GCC TTC TAG-3' and 5'-TAG CAG CTT TCC CCA GCA TGC CTG CTA-3', and then fused to the yCD cDNA by overlap PCR. The entire 702-bp fragment carrying yCD and poly(A) was subcloned into pAdMCSloxp on a HindIII/HindIII site to yield pAdyCDloxp. The integrity of the sequence was verified by sequencing.

The CEA basal promoter was isolated by PCR from human genomic DNA (Boehringer Mannheim, Mannheim, Germany). The 5' primer (5'-GCG CTC TAG AGC GGC CGC CCC GGG ACC CTG CTG GGT TTC-3') was designed to have restriction sites for XbaI and NotI. The 3' primer (5'-GCG CTC TAG AAA GCT TGA GTT CCA GGA ACG-3') contained restriction sites for Xba and HindIII. The resulting 368-bp fragment cloned into the Not/Xba site upstream from the yCD in pAdyCDloxp yielded pAdloxpCEA-basal-yCD. The enhancer sequence of CEA was then amplified in two fragments using overlap PCR with oligonucleotides (5'-GCG CGC GGC CGC TCT AGA GGT TAC ATT ACA AAG TGG AAT G-3' and 5'-TTA AAG GGA GAA AAG ACA ATA CA-3') and (5'-GGA GTG CCC TTC AGT CAA TA-3' and 5'-GCG CGC GGC CGC TCTA GAC GGC TCA CTG CAA CCT CTG CCT C-3') to give the 5' and 3' fragments, respectively. These were then fused together by PCR before subcloning into pAdloxpCEA-basal-yCD. This yielded pAdloxpCEA-basal-enhancer-yCD (Fig. 1)Citation .



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  		Fig. 1. Diagrammatic representation of constructs used for the study. The CEA basal promoter was isolated by PCR from human genomic DNA from -266 and +102 of the transcriptional start site of CEA. The resulting 368-bp fragment was cloned upstream from the yCD in pAdyCDloxp yielding pAdloxpCEA-basal-yCD. The enhancer sequence of CEA was then amplified by PCR from -6.1 to -4.0 before subcloning into pAdloxpCEA-basal-yCD to construct pAdloxpCEA-basal-enhancer-yCD.

 
Transfection
To assess yCD activity, pAdRSVyCD, pAdCEAyCD, and pAdCEA-basal-enhancer-yCD were transfected into LoVo and MCF-7 cell lines, and normal HFs using LipofectAMINE Plus (Life Technologies, Inc., Rockville, MD) following the manufacturer’s instructions. Cell extracts were prepared 48 h after transfection to analyze yCD activity.

Ad Production and Titration
Adenoviral constructs were generated by in vitro recombination. In brief, the yCD was inserted in an adenoviral shuttle plasmid (pAdMCSloxP) containing either a RSV- or CEA-driven expression cassette. These were used to generate a replication-defective adenoviral genome by cre/loxP recombination with a cosmid, cs360loxP, containing most of the Ad type-5 backbone. Viruses were generated by transfection into the 293-complementation cell line. The resulting adenoviral constructs contain the dl309 E3 deletion, whereas the E1 region was replaced by the expression cassette containing, from 5' to 3', the RSV or CEA promoter-enhancer. Virus was propagated in 911 cells and purified on a CsCl gradient. Purified viruses were stored in 10 mM Tris-HCl (pH 7.4), 137 mM NaCl, 5 mM KCl, and 1 mM MgCl2 in 10% glycerol (by volume).

Ad Infections in Vitro
All of the infections were performed by incubating cells for 3 h with Ad particles diluted in 100 µl of culture media supplemented with 2% FBS at 37°C. Cells were then plated into complete medium and analyzed at various times after infection. To correct for adenoviral infectivity, we first determined the percentage of cells infected with a CMV-driven green fluorescent protein Ad at 100 MOI using fluorescence microscopy. We found that SCCVII and HF cells were infected much less readily than the other cell lines, presumably because of a lack of Coxsackie adenovirus receptor receptor (24 , 25) . Therefore, to produce infection in the same general range for all of the cell lines, we used 500 MOI for SCCVII and HF and 100 MOI for the other cell lines. CD enzyme activity was then normalized to UMSCC6 cells, which showed the highest infection rate (69%).

RNA Isolation and RT-PCR
Cells were harvested in TRIzol (Invitrogen, San Diego, CA) and passed through a 28-gauge needle-fitted syringe to shear DNA. RNA was extracted and purified by phenol-chloroform, precipitated with isopropanol, dissolved in RNase-free water, and stored at -20°C until used. RNA (1 µg) was reverse transcribed to cDNA under standard conditions. This cDNA product was then used for amplification of CEA by PCR reactions. CEA-specific primer used in this study were: 5'-CGC CAA AAT CAC GCC AAA TAA TAA-3' and 5'-ACC CCA ACC AGC ACT CCA ATC AT-3' (product size, 171 bp). After PCR amplification, the product was separated on a 2% (w/v) agarose gel in TAE buffer [Tris, 40 mM; Na2 EDTA, 1 mM; Na Acetate, 40 mM (pH 8)], stained with 0.5 µg/ml ethidium bromide, and photographed under UV light. GAPDH transcripts were used to control for the RNA that was used to amplify the genes by RT-PCR.

Immunoblotting
Proteins were extracted in 1% NP40 lysis buffer and were resolved on a 4–20% gradient GradiGels (Gradipore Ltd., Frenchs Forest, Australia). After blotting, the membrane was blocked with 5% nonfat dried milk in Tris-buffered saline containing 0.1% Tween 20, followed by a 2-h incubation with either rabbit anti-yCD serum (1:50000; custom made by Berkeley Antibody Company, Richmond, CA) or CEA Ab3 (1:5000). The secondary horseradish peroxidase-labeled goat antirabbit or antimouse IgG (1:10000; Southern Biotechnology Inc., Birmingham, AL) was then added for 2 h. The proteins were visualized using chemiluminescence (Pierce, Rockford, IL).

Enzyme Activity Assay
yCD activity was quantified by the percentage of conversion (6-[3H]) 5-FC as described previously (12) . We established a linear correlation (r2 = 0.99) between purified yCD enzyme used (1.6–200 ng) and 5-FC to 5-FU conversion activity (0.32–79%) detected in this assay (Fig. 2)Citation . For in vitro or in vivo experiments, cell extracts were made by four to five 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 yCD activity assay, either 100 ng of purified yCD as positive control, or 30 µg of cell extract, or 100 µg of tumor homogenate were incubated with 0.5 mM (6-[3H]) 5-FC in a 30-µl-reaction volume overnight at 37°C. The produced (6-[3H]) 5-FU was isolated by elution from a SCX Bond Elute column (Varian, Harbor City, CA), counted, and expressed as a fraction of the total counts.



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  		Fig. 2. Correlation between 5-FC to 5-FU conversion and quantity of yCD. Known quantities of purified yCD enzyme were assessed for 5-FC to 5-FU converting activity as described in Materials and Methods. A linear correlation (r2 = 0.99) between the yCD enzyme used (1.6–200 ng) and 5-FC to 5-FU conversion activity (0.32–79%) was demonstrated.

 
Tumor Xenograft Models
Intrahepatic Tumors.
Female, 7–8-week-old, Nu/Nu CD-1 nude mice (Charles River Laboratories, Wilmington, MA) were handled according to the established procedures of the University of Michigan Laboratory Animal Maintenance Manual. Mice were anesthetized with an i.p. injection of 9 mg/kg xylazine (Bayer Co., Kansas City, MO) and 58 mg/kg ketamine (Fort Dodge Laboratories Inc., Fort Dodge, IA). The left liver lobe was exteriorized through a 1-cm midline incision and held using sterile cotton-tipped swabs. Approximately 106 HT-29 cells were injected into the liver lobe, the peritoneal wall was sutured (Ethicon, Somerville, NJ), and the body wall was closed by autoclips (MikRon Precision, Gardena, CA). Ten days after tumor implantation, animals were divided into three groups for a single i.v. injection of 1010 pfu of empty virus (group I), RSV yCD virus (group II), or enhanced CEA yCD (group III). Two days after the virus injection, mice were autopsied. The intrahepatic tumor and other tissues were then processed for yCD enzyme activity.

s.c. Tumors.
s.c. tumor xenografts were established by injecting 5 x 106 viable cells (in 100 µl of PBS) s.c. into the flanks of 8-week-old mice. Tumors were measured with calipers in 2 dimensions. Tumor volumes were calculated using the formula ({pi}/6)(L x W2), where L = length of tumor and W = width. When tumors measured an average volume of 100 mm3, mice were randomized into three groups (6–10 mice/group), and the treatment was initiated. A total of 1 x 109 pfu of AdCEAyCD, AdRSVyCD, or empty virus was injected into the tumors on days 0, 4, 7, and 10. 5-FC (500 mg/kg) or PBS was given i.p. on days 3–7 and 10–14, 3 h before radiation treatment (3 Gy to the combination-therapy group), based on our magnetic resonance spectroscopy data that 5-FC conversion to 5-FU is maximal at that time (13) . Mice were killed on day 60, or sooner if the tumor volume exceeded 10 times the volume at day 0.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
To evaluate the specificity of the CEA promoter regions, it was first necessary to identify and rank CEA-expression levels in different cell lines (Fig. 3)Citation . Human colon cancer cell lines LoVo, HT-29, NCI-H508, and Caco-2 and human pancreatic cell-line BxPC-3 were found to produce high levels of both CEA protein and mRNA as detected by immunoblot and RT-PCR analysis. Human breast carcinoma MCF-7 cells had lower but detectable levels of CEA. On the other hand, murine head and neck carcinoma cells SCCVII, human head and neck carcinoma UMSCC6, neonatal HF, and hepatoma HuH-7 did not produce detectable CEA by either analysis.



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  		Fig. 3. Analysis of CEA transcript and protein levels in different cell lines. RNA transcripts were determined by RT-PCR (A and B) and protein content by immunoblotting (C and D), as described in "Materials and Methods." A, CEA transcript; B, GAPDH loading control; C, CEA protein; D, Actin loading control. KDa, Mr in thousands.

 
Comparison of RSV-, CEA Basal-, and CEA Promoter-Enhancer in Vitro.
We selected three cell lines with various degrees of CEA levels (LoVo, MCF-7, and HF) for transient transfection to evaluate the CEA promoter’s efficiency and selectivity. We first determined that these three cell lines showed similar levels of yCD expression from pAdRSVyCD on transfection. We then assessed the efficacy of the basal CEA promoter. As anticipated, although the CEA promoter conferred specificity of expression to CEA-producing LoVo cells, the activity was very low compared to that with the RSV promoter (Fig. 4)Citation . To attempt to enhance the CEA basal promoter activity, we incorporated a 2.1-kb CEA enhancer sequence from -4.0 to -6.1 of the transcription start site upstream to the basal CEA promoter (Fig. 1)Citation . We found that there was an almost 30-fold increase in expression in the strongly CEA-expressing LoVo cells, an 8-fold increase in the moderately expressing MCF-7 cells, but no increase in yCD expression in human skin fibroblasts. Indeed, expression in LoVo cells by the enhanced CEA exceeded that produced by pAdRSVyCD.



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  		Fig. 4. Expression of yCD under RSV or CEA promoters after transient transfection, Cells were transfected with one of three vectors: pAdRSV-yCD, pAdCEA-basal-yCD, or pAdCEA-enhancer-yCD. Twenty-four h after transfection, cells were harvested, and the lysates were assessed for yCD activity by measuring the conversion of 5-FC to 5-FU.

 
Comparison of RSV and CEA Promoter-Enhancer in Vitro.
On the basis of these encouraging transient transfection experiments, we constructed recombinant replication-defective Ad vectors, pAdloxpRSVyCD and pAdloxpCEA-enhancer-yCD (Ad vector with yCD driven by either RSV or CEA promoter-enhancer), as described in "Materials and Methods." For this purpose we used 10 different cell lines with various extents of CEA expression (Fig. 3)Citation . We found that there was a strong relationship between yCD expression driven by CEA promoter-enhancer and CEA expression levels (corrected for infectivity as described in "Materials and Methods"; Fig. 5Citation ). Importantly, the yCD activity obtained from CEA promoter-enhancer in various CEA-expressing cell lines was at least as high as that of RSV (Fig. 6)Citation .



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  		Fig. 5. Analysis of yCD expression achieved from AdCEA-yCD and AdRSV-yCD infection. Cells were infected with either AdCEA-enhancer-yCD (A) or AdRSV-yCD (C) as described in "Materials and Methods." Twenty-four h after infection, cell lysates were immunoblotted and probed with anti-yCD rabbit serum. To demonstrate equal loading, the blots were stripped and reprobed with antibodies against ß-actin (B and D).

 


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  		Fig. 6. Comparison of CEA-yCD and RSV-yCD enzyme activity. Cells were infected with either AdRSV-yCD or AdCEA-enhancer-yCD as described in "Materials and Methods." Twenty-four h after infection, cells were assessed for yCD activity by 5-FC to 5-FU conversion. Data are expressed as the ratio of yCD activity from AdCEA-enhancer-yCD compared with that from AdRSV-yCD.

 
Comparison of RSV and CEA Promoter-Enhancer in Vivo.
It was next of interest to determine whether the selectivity and activity found in vitro could occur in vivo. To assess this question, we injected HT-29 colon cancer cells into the livers of nude mice; and after 2 weeks, animals were divided into three groups of six animals each. Group I received only saline, group II received AdRSV-yCD, and group III received AdCEA-yCD i.p. Normal tissues and tumor cells were harvested 48 h after the Ad injection. We found negligible yCD activity in colon, blood, muscles, bone marrow, liver, spleen, and skin. Specific Ad-mediated in vivo transduction of the yCD gene was shown selectively only in CEA-producing HT-29 cells growing in the liver. The expression of yCD was 2-fold greater than that produced by the RSV-yCD Ad. The enzyme activity from tumor cells was 14 times higher than that of the normal liver (Fig. 7)Citation .



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  		Fig. 7. Comparative analysis of yCD expression in different organs of nude mice after RSV- or CEA-driven yCD-Ad injection. Intrahepatic tumors were prepared as described in "Materials and Methods." After confirmation of the presence of tumor, RSV- and CEA-driven yCD-Ad were injected i.p. Seventy-two h later, the animals were killed, and the organs were assessed for yCD expression.

 
To determine whether the efficacy of AdCEA-yCD and AdRSV-yCD were similar in vivo, we assessed their effect in athymic mice carrying a s.c. HT-29 flank tumor. Ad carrying the yCD gene under the CEA promoter-enhancer or the RSV promoter were injected into the tumor on days 0, 4, 7, and 10. 5-FC injections were given on days 0–4 and 7–11 with or without radiation. We found that the two adenoviral vectors produced no effect when administered alone, but that both produced similar growth inhibition when animals received 5-FC with or without radiation (Fig. 8)Citation .



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  		Fig. 8. Effect of 5-FC treatment with or without radiation on the growth of HT-29 flank tumor in nude mice. HT-29 flank tumors were prepared as described in "Materials and Methods." When tumors reached 100 mm3, RSV-driven (A) or CEA promoter-enhancer-driven (B) yCD Ad was injected into the tumor on days 0, 4, 7, and 10. On days 0–4 and 7–11, animals were treated with 5-FC (500 mg/kg; {blacksquare}; n = 6), radiation (3 Gy x 10; {bullet}; n = 12), or a combination of 5-FC and radiation ({diamondsuit}; n = 10), as described in "Materials and Methods." Tumor growth after administration of saline alone ({blacktriangleup}; n = 18) or with vector ({blacktriangledown}; n = 6) is also shown. The average volumes (±SE) relative to those at day 0 are shown. Horizontal bar, the treatment period.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
For gene therapy to be effective in the treatment of colorectal carcinoma, it is essential to target tumor cells while sparing normal tissues. In this study, we have found that the transduction of the yCD gene under the transcriptional control of the CEA promoter-enhancer confers selectivity of yCD expression to CEA-producing cells without a loss of activity. To enhance the low level of activity of the basal promoter, a 2.1-kb enhancer sequence upstream from the CEA promoter (-6.1 to -4.0 from the transcriptional start) was inserted. This approach led to a 30-fold higher expression of yCD than was produced by the CEA promoter alone, with an overall expression level in CEA-positive cells that is comparable with that induced by the active but nonspecific RSV promoter. Specificity and selectivity were shown in vitro using transient transfection and infection with Ad. Specificity for tumor was also demonstrated by selective expression of yCD in CEA-expressing cells implanted in the liver after systemic administration of virus, and activity was shown in therapy studies.

Tumor-specific promoters have been shown to be selective tools for targeting tumor cells in various studies (21 , 23) . Although these reports have demonstrated selective expression of CD or herpes simplex virus-thymidine kinase (HSV-TK) in tumor cells using a selective promoter-enhancer scheme, it has been at the cost of overall activity (23 , 26) . For instance, Kanai et al. (23) constructed selective AdAFPCD, which they compared with a nonselective Ad-CMV-immediate-early enhancer-chicken ß-actin hybrid-CD (AdCAGCD). They found that the CD expression from AFP promoter-enhancer was about 1/300th that of the nonspecific chicken-ß-actin promoter (22) . Richards et al. (27) have reported that multimerization of a part of the CEA promoter, alone or fused with its specific enhancer sequences, resulted in copy number-related increases in promoter activity. A previous attempt to use a CEA enhancer sequence upstream from the CEA promoter sequence did not produce a significant increase in the reporter chloramphenicol acetyl transferase (CAT) gene activity as analyzed by CAT assay (28) .

Although we were encouraged to see selectivity of expression in CEA-expressing liver tumors in the nude mouse studies, we recognize that additional work will be required to establish specificity in patients. Because the promoters constructed in our Ad vectors are derived from human genomic DNA, there is a possibility that activation of these elements may not occur in normal tissues of nude mice. To have addressed this more rigorously, it would have been necessary to design an enhanced mouse CEA promoter, which would not have been applicable to human studies. It is also important to note that a small amount of CEA is produced by some human tissues, such as antral mucosa and bile ducts (29) . Although our studies suggest that a small amount of CEA is unlikely to drive substantial yCD production, the potential toxicity against normal human tissues needs to be assessed in carefully conducted toxicity trials when these vectors are used in patients.

Although our experiments were aimed at comparing promoter strategies rather than achieving optimal tumor response, our data suggest that it will be important to increase enzyme expression. For instance, an active CEA-selective promoter may be attractive to consider with replication-competent virus (30) , because the toxicity of nonselective infection of normal tissues should be minimized by selective expression. In addition, it may be possible to manipulate regional infusions of the portal vein or hepatic artery to increase tumor delivery.4 It seems likely that a combination of approaches will need to be used to make gene therapy successful.


   ACKNOWLEDGMENTS
 
We thank Daniel A. Hamstra and Mary Davis for help throughout this work and Michael Peacock, Vector Core Lab, University of Michigan, for Ad generation.


   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 Supported by NIH Grant CA80145 and Cancer Center Core Grant CA46592. Back

2 To whom requests for reprints should be addressed, at Department of Radiation Oncology, University of Michigan, 1500 East Medical Center Drive, UH-B2C490, Ann Arbor, MI 48109-0010. Phone: (734) 647-9955; Fax: (734) 763-7371; E-mail: tsl{at}umich.edu Back

3 The abbreviations used are: 5-FC, 5-fluorocytosine; 5-FU, 5-fluorouracil; CD, cytosine deaminase; yCD, yeast CD; CEA, carcinoembryonic antigen; CMV, cytomegalovirus; RSV, rous sarcoma virus; AFP, {alpha}-fetoprotein; Ad, adenovirus; HF, human fibroblast; MOI, multiplicity/multiplicities of infection; (poly)A, polyadenylic acid; RT-PCR, reverse transcription-PCR; pfu, plaque-forming unit(s); GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Back

4 M. Zhang, S. P. Li, M. K. Nyati, S. DeRemer, J. Parsels, A. Rehemtulla, W. D. Ensminger, and T. S. Lawrence. Adenovirus mediates a selective expression of yeast cytosine deaminase in intrahepatic colon carcinoma via CEA promoter, manuscript in preparation. Back

Received 11/19/01. Accepted 2/ 5/02.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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