Stable expression of a GFP-BSD fusion protein in Babesia bovis merozoites.

Transfection has been a valuable technique for elucidating gene function in many pathogens. While transient transfection of Babesia spp. has been reported previously, stable integration of exogenous genes in Babesia has proven difficult. In this study, a plasmid was designed to target integration of a gfp-bsd gene into the Babesia bovis ef-1alpha locus. Babesia bovis-infected erythrocytes of the biologically cloned Mo7 strain were transfected by electroporation with either circular or linear plasmids and selected in cultures with varying amounts of blasticidin 24h after electroporation. Several blasticidin-resistant B. bovis transfected cell lines emerged at different rates, ranging from 5 to 26 days after the start of selection. One transfected parasite line (1-2-124) was selected for further analysis based on a rapid growth rate and bright GFP fluorescence in the presence of a lethal concentration of blasticidin. Continued expression of the gfp-bsd fusion gene was confirmed by reverse transcriptase-PCR, Western blot analysis and fluorescence microscopy for longer than 9 months after electroporation. No plasmid or episomal DNA could be detected in this line, and plasmid recovery in Escherichia coli was unsuccessful. Southern blot results and sequencing of PCR amplicons flanking the putative insertion site are consistent with integration of at least one gfp-bsd cassette into the targeted ef-1alpha locus in the transfected parasite line. Overall the results demonstrate, we believe for the first time, chromosomal integration and stable expression of a foreign gene in B. bovis. With the availability of the B. bovis genome, targeted stable transfection will provide a means to determine the role of specific genes in the biology, clinical disease and immunity of B. bovis, one of the three major tick-borne parasites that limit global livestock production.

Expression of APOBEC3G in kidney cells.

The apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G (APOBEC3G), a member of the APOBEC family possessing DNA mutator activity through cytosine deamination, is reported to play an important role in host defense against infections such as those of hepatitis B virus and human immunodeficiency virus. Here, we examined the expression of APOBEC3G in human kidney cells to better understand its biological role against infection. APOBEC3G was immunohistochemically detectable in kidney mesangial cells and also to some extent in kidney epithelial tubular cells. In addition, overexpression of APOBEC3G was shown in renal carcinoma tissues and cell lines. APOBEC3G expression was upregulated by inflammatory cytokines, such as interferon, interleukin-6, and tumor necrosis factor. These results may provide new insight into the role of APOBEC3G in host defense against viral infection and cancer.

Distinct patterns of cytokine regulation of APOBEC3G expression and activity in primary lymphocytes, macrophages, and dendritic cells.

Human APOBEC3G (A3G), a deoxycytidine deaminase, is a broadly acting antiretroviral factor expressed in a variety of cells. Mitogen activation of CD4 T cells enhances A3G expression and leads to recruitment of low molecular mass (LMM) A3G, which functions as a post-entry human immunodeficiency virus (HIV) restriction factor, into enzymatically inactive, high molecular mass (HMM) RNA-protein complexes that include Staufen RNA-transporting granules. We now report that interleukin-2 (IL-2), IL-15 and, to a lesser extent, IL-7 enhance the expression of A3G in peripheral blood lymphocytes and that this effect is blocked by inhibitors of the JAK and MAPK signaling pathways. In mixed cultures of CD4+ T cells containing either HMM or LMM A3G, HIV preferentially infected cells containing HMM A3G. A3G shifted into a HMM complex when IL-2, -7, or -15 was added to resting T cells, likely explaining how cytokine treatment renders resting CD4+ T cells permissive to HIV infection. Similarly, poly(I:C)/tumor necrosis factor-alpha-induced maturation of dendritic cells was associated with a sharp increase in A3G expression; however, this induction led to the accumulation of LMM A3G. Together, these results highlight the distinct inductive effects of select cytokines on A3G gene expression and A3G complex assembly that occur in natural cellular targets of HIV infection.

In vivo induction of resistance to gemcitabine results in increased expression of ribonucleotide reductase subunit M1 as the major determinant.

Gemcitabine is a deoxycytidine (dCyd) analogue with activity against several solid cancers. Gemcitabine is activated by dCyd kinase (dCK) and interferes, as its triphosphate dFdCTP, with tumor growth through incorporation into DNA. Alternatively, the metabolite gemcitabine diphosphate (dFdCDP) can interfere with DNA synthesis and thus tumor growth through inhibition of ribonucleotide reductase. Gemcitabine can be inactivated by the enzyme dCyd deaminase (dCDA). In most in vitro models, resistance to gemcitabine was associated with a decreased dCK activity. In all these models, resistance was established using continuous exposure to gemcitabine with increasing concentrations; however, these in vitro models have limited clinical relevance. To develop in vivo resistance to gemcitabine, we treated mice bearing a moderately sensitive tumor Colon 26-A (T/C = 0.25) with a clinically relevant schedule (120 mg/kg every 3 days). By repeated transplant of the most resistant tumor and continuation of gemcitabine treatment for >1 year, the completely resistant tumor Colon 26-G (T/C = 0.96) was created. Initial studies focused on resistance mechanisms known from in vitro studies. In Colon 26-G, dCK activity was 1.7-fold decreased; dCDA and DNA polymerase were not changed; and Colon 26-G accumulated 1.5-fold less dFdCTP, 6 hours after a gemcitabine injection, than the parental tumor. Based on in vitro studies, these relative minor changes were considered insufficient to explain the completely resistant phenotype. Therefore, an expression microarray was done with Colon 26-A versus Colon 26-G. Using independently grown nonresistant and resistant tumors, a striking increase in expression of the RRM1 subunit gene was found in Colon 26-G. The expression of RRM1 mRNA was 25-fold increased in the resistant tumor, as measured by real-time PCR, which was confirmed by Western blotting. In contrast, RRM2 mRNA was 2-fold decreased. However, ribonucleotide reductase enzyme activity was only moderately increased in Colon 26-G. In conclusion, this is the first model with in vivo induced resistance to gemcitabine. In contrast to most in vitro studies, dCK activity was not the most important determinant of gemcitabine resistance. Expression microarray identified RRM1 as the gene with the highest increase in expression in the Colon 26-G, which might clarify its complete gemcitabine-resistant phenotype. This study is the first in vivo evidence for a key role for RRM1 in acquired gemcitabine resistance.

Regional delivery and selective expression of a high-activity yeast cytosine deaminase in an intrahepatic colon cancer model.

A major potential limitation to the success of enzyme prodrug gene therapy is the toxicity that could result from gene expression in normal tissues. In this study, we investigated the use of an enhanced human carcinoembryonic antigen (CEA) promoter for yeast cytosine deaminase (yCD), which converts 5-fluorocytosine to 5-fluorouracil, to increase targeting while maintaining activity both in cell culture and in nude rats bearing intrahepatic xenografts. We found that an enhanced CEA-yCD adenoviral vector can achieve significantly greater yCD expression in CEA-expressing colon carcinoma cell lines (LoVo, HT29, and CaCo2) compared with a nonspecific Rous sarcoma virus-yCD virus. In contrast, infection with CEA-yCD led to lower or equivalent yCD expression in normal hepatocytes or fibroblasts compared with that produced by the RSV-yCD. Adenovirus administered in the portal vein or the hepatic artery of nude rats bearing intrahepatic LoVo colon carcinomas could mediate beta-galactosidase expression equally in liver and tumors under the control of cytomegalovirus, a nonspecific promoter. However, infusion of CEA-yCD virus markedly increased yCD expression in tumors over normal liver (>4-fold) measured both by levels of mRNA and yCD activity. Moreover, the efficiency of 5-fluorocytosine conversion into 5-fluorouracil in tumors was significantly higher than that in normal liver ( approximately 3-fold) in rats receiving portal venous viral infusion of CEA-yCD and subsequent 5FC treatment. Thus, an enhanced CEA promoter can preferentially stimulate yCD gene expression in CEA-expressing cells in vivo. Such tumor-specific expression should prove useful in colorectal cancer gene therapy to achieve selective prodrug conversion in tumors.

Cancer-specific killing by the CD suicide gene using the human telomerase reverse transcriptase promoter.

Human telomerase reverse transcriptase (hTERT), the catalytic subunit of the telomerase, is transcriptionally upregulated in more than 90% of tumor cells. It may be used as a tool for driving a gene to kill tumors specifically. To test this idea, luciferase reporter gene was used and the results showed that hTERT promoter could restrict the gene expression in the telomerase-positive tumor cells. A tumor-specific expression plasmid phTERT-CD was constructed, in which the E. coli cytosine deaminase (CD) gene was controlled by the hTERT promoter. A colorectal cancer cell line (LoVo) and a normal amnion cell line (WISH) were transfected by this plasmid. It was shown that the expression of the CD gene increased the sensitivity of LoVo cells to the prodrug, 5-fluorocytosine (5FC), over 800-fold, while the sensitivity of WISH cells to 5FC was increased only 6-fold. Mixed cell experiments showed a strong "bystander effect" on CD-negative cells. Furthermore, a significant anti-tumor effect of the phTERT-CD/5FC system was observed in nude mice bearing mammalian carcinoma induced by s.c. inoculation of LoVo cells when the mice were given 250 mg/kg 5FC twice a day for 10 consecutive days. These results indicated that hTERT promoter could target the suicidal effect of CD gene to tumor cells, and therefore, may be a novel and promising targeting approach to the treatment of cancer.

High and selective expression of yeast cytosine deaminase under a carcinoembryonic antigen promoter-enhancer.

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.

Quantitation of cytosine deaminase mRNA by real-time reverse transcription polymerase chain reaction: a sensitive method for assessing 5-fluorocytosine toxicity in vitro.

Cytosine deaminase/5-fluorocytosine (CD/5-FC) is a promising strategy for local cancer gene therapy. We hypothesized that CD expression within tumor cells would be directly related to efficacy and that quantitation of markers of CD expression such as mRNA, protein, and enzyme activity would therefore facilitate prediction of 5-FC toxicity. These three markers were thus quantitated by real-time quantitative reverse transcription polymerase chain reaction (Q-RT-PCR), semiquantitative immunocytochemistry (ICC), and 5-[(3)H]FC enzyme assay, respectively. Results with human colon (LS174T) cancer cells infected with a replication-incompetent adenovirus encoding CD (AdCMVCD) demonstrated a significant correlation between CD mRNA and enzyme activity up to 24 h postinfection. A direct correlation was found between CD dose (AdCMVCD PFU/cell) and CD mRNA and protein expression (P < 0.002) in both LS174T and BxPC-3 pancreatic cancer cells, but the relationship with enzyme activity was less strong in LS174T cells (P = 0.09). A remarkable concordance existed among Q-RT-PCR, ICC and enzyme assays with both cell lines. Importantly, CD dose and mRNA and protein expression inversely correlated with 5-FC IC(50) (P < 0.02). Quantitation of CD markers also facilitated identification of factors governing differential susceptibility to CD/5-FC. These results suggest that Q-RT-PCR will be useful for monitoring transgene expression in future studies using improved CD-based expression vectors and may also be useful in predicting the response to CD/5-FC therapy, which is likely to be heterogeneous in the patient population.

A phase I trial of genetically modified Salmonella typhimurium expressing cytosine deaminase (TAPET-CD, VNP20029) administered by intratumoral injection in combination with 5-fluorocytosine for patients with advanced or metastatic cancer. Protocol no: CL-017. Version: April 9, 2001.

An attenuated strain of Salmonella typhimurium, designated VNP20009, was generated by deletion of the msbB and purl genes. When VNP20009 was administered intravenously (IV) to mice bearing spontaneous, syngeneic, or human xenograft tumors, the bacteria accumulated preferentially within the extracellular components of tumors, forming tumor-to-normal tissue ratios exceeding 300-1000 to 1. NVP20009 was administered safely at doses up to 2.5 x 10(9) cfu/kg in monkey toxicology studies. Based on the preclinical data, VNP20009 entered Phase I human clinical trials in November 1999, and has now been administered to >45 patients by IV or direct intratumoral injection. By the intratumoral route, a maximum tolerated dose has not been reached, and dose escalation continues past the current dose level of 4 x 10(7)/m2. Furthermore, VNP20009 persisted in injected tumors for at least 2 weeks in 8/11 patients treated to date. By 30-min IV administration, a maximum tolerated dose (MTD) of 3 X 10(8) cfu/m2 has been established. In all patients treated to date, VNP20009 was not shed in urine or stool. VNP20009 has been further modified by chromosomal insertion of an E. coli cytosine deaminase (CD) gene at the deltamsbB locus which, when expressed, converts 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU). The CD containing VNP20009 was designated TAPET-CD or VNP20029. TAPET-CD had similar efficacy and safety in murine tumor models and similar safety profiles in animal toxicology studies, compared to its parent VNP20009. Specifically, TAPET-CD had a reduced virulence of >10,000 fold, when compared to the wild-type Salmonella strain. It was well-tolerated at doses up to 2 x 10(6) cfu/mouse and 1 X 10(10) cfu/monkey. After an IV or direct tumor injection to tumor-bearing mice, TAPET-CD reached tumor levels as high as 10(8)-10(9) cfu/gm. When compared to the accumulation in liver or spleen, the normal tissues with the greatest colonization of TAPET-CD, tumor-to-normal tissue ratios of TAPET-CD were 300-1000 to 1. TAPET-CD also caused tumor growth inhibition of >90% in several murine tumor models. When 5-FC was administered by intraperitoneal (IP) injection once or 3 times daily to tumor-bearing mice that had been pre-treated with TAPET-CD, high levels of 5-FU (reaching 20-40 microM/g) were detected in the tumor, with low or undetectable 5-FU levels in normal tissues (e.g., spleen, liver, etc.). Furthermore, co-administration of 5-FC and TAPET-CD in 4 different murine tumor models enhanced anti-tumor activity compared to the significant anti-tumor activity of TAPET-CD alone, further confirming the benefit of the inserted CD gene. On the basis of the preclinical data, a Phase I clinical protocol is proposed in which advanced cancer patients will receive TAPET-CD by direct intratumoral injection and 5-FC. TAPET-CD will be administered on day 1. 5-FC will be given orally q8h daily beginning day 4 or when all toxicities of TAPET-CD have resolved to < or = grade 1, and continued for 14 days. Tumor tissues will be sampled to verify TAPET-CD colonization and to measure intratumoral 5-FC and 5-FU concentrations on day 8. A second sample of tumor tissue will be obtained between day 15-17 in selected patients to confirm the persistence of high levels of bacteria in tumor and to obtain a second measurement of 5-FC and 5-FU intra-tumoral concentrations. The TAPET-CD/5-FC treatment cycle will be repeated in appropriate patients on day 29.

The use of the L-plastin promoter for adenoviral-mediated, tumor-specific gene expression in ovarian and bladder cancer cell lines.

A 2.4-kb truncated L-plastin promoter was inserted either 5' to the LacZ gene (Ad-Lp-LacZ) or 5' to the cytosine deaminase (CD) gene (Ad-Lp-CD) in a replication-incompetent adenoviral vector backbone. Infectivity and cytotoxicity experiments with the LacZ and CD vectors suggested that the L-plastin promoter-driven transcriptional units were expressed at much higher levels in explants of ovarian cancer cells from patients and in established ovarian or bladder cancer cell lines than they were in normal peritoneal mesothelial cells from surgical specimens, in organ cultures of normal ovarian cells, or in the established CCD minimal deviation fibroblast cell line. Control experiments showed that this difference was not attributable to the lack of infectivity of the normal peritoneal cells, the normal ovarian cells, or the minimal deviation CCD fibroblast cell line, because these cells showed expression of the LacZ reporter gene when exposed to the replication-incompetent adenoviral vector carrying the cytomegalovirus (CMV)-driven LacZ gene (Ad-CMV-LacZ). The Ovcar-5 and Skov-3 ovarian cancer cell lines exposed to the Ad-Lp-CD adenoviral vector were much more sensitive to the prodrug 5-fluorocytosine (5FC), which is converted from the 5FC prodrug into the toxic chemical 5-fluorouracil, than was the CCD minimal deviation fibroblast cell line after exposure to the same vector. A mouse xenograft model was used to show that the Ad-Lp-CD vector/5FC system could prevent engraftment of ovarian cancer cells in nude mice. Finally, injection of the Ad-Lp-CD vector into s.c. tumor nodules generated a greater reduction of the size of the tumor nodules than did injection of the Ad-CMV-LacZ vectors into tumor nodules. The Ad-Lp-CD vectors were as suppressive to tumor growth as the Ad-CMV-CD vectors. These results suggest that an adenoviral vector carrying the CD gene controlled by the L-plastin promoter (Ad-Lp-CD) may be of potential value for the i.p. therapy of ovarian cancer.

Usefulness of repeated direct intratumoral gene transfer using hemagglutinating virus of Japan-liposome method for cytosine deaminase suicide gene therapy.

To investigate the feasibility of repeated gene transfection in suicide gene therapy against human solid tumors by a combination of 5- fluorocytosine (5-FC) and its converting enzyme, cytosine deaminase (CD), we repeatedly transfected the yeast CD gene into the human pancreatic cancer cell line BXPC3 using the hemagglutinating virus of Japan-liposome in a new gene transfer method. The in vivo growth of the s.c. transplanted BXPC3 tumor in nude mice given CD-gene transfection was significantly suppressed by i.p. injection of 5-FC when compared with tumors treated with the control vector. Furthermore, the tumor transfected with the CD gene during a 7-day interval was suppressed much more than that of a single transfection. These results suggest that repeated transfection of the suicide gene together with the combination of 5-FC and the yeast CD gene using the hemagglutinating virus of Japan-liposome gene transfer method may be useful for the treatment of human solid tumors, including pancreatic cancer.

Breast cancer-specific expression of the Candida albicans cytosine deaminase gene using a transcriptional targeting approach.

We constructed a series of adenoviral (Ad) vectors that express the Candida albicans cytosine deaminase (CD) suicide gene under the transcriptional control of either the human alpha-lactalbumin (ALA) or ovine beta-lactoglobulin (BLG) promoter (Ad.ALA.CD and Ad.BLG.CD, respectively). The Ad.ALA.CD and the Ad.BLG.CD vectors converted the prodrug 5-fluorocytosine (5-FC) to the toxic nucleotide analog 5-fluorouracil in a breast cancer cell-specific manner, with a conversion rate of 40% and 52% in T47D cells and 50% and 41% in MCF7 cells, respectively. No significant conversion (< or =3%) was observed in an immortalized nontumorigenic breast epithelial cell line (MCF10A) and a human osteosarcoma cell line (U2OS). Adenovirus vector-based prodrug conversion of the 5-FC in T47D and MCF7 in the presence of 1 mg/mL of 5-FC led to cytotoxicity that resulted in a nearly complete cell death (> or =90%) after 5 days, whereas MCF10A and U2OS cells remained resistant (< or =10%). Nude mice harboring T47D-derived breast tumors that were injected intratumorally (i.t.) with therapeutic adenovirus vectors at a dose of 2 x 10(8) plaque-forming units and treated systemically with 5-FC at a concentration of 500 mg/kg/day showed a marked reduction in tumor mass within 30 days when compared with animals that received vector alone. Animal survival was significantly prolonged after 72 days in mice treated with therapeutic vectors in conjunction with prodrug when compared with control animals. These preclinical data are sufficiently promising to warrant further studies of this transcriptional targeting approach to breast cancer treatment.

Gene therapy of metastatic colon carcinoma: regression of multiple hepatic metastases by adenoviral expression of bacterial cytosine deaminase.

Colon carcinoma accounts for 20% of deaths due to malignancies in the Western world. Once metastases occur, therapeutic options are limited, with an approximate 5-year survival of only 5%. To investigate the potential of new gene therapeutic approaches, a hepatic micrometastasis model of colon carcinoma in BALB/c mice was established. Inoculation of syngeneic MCA26 colon carcinoma cells into the spleens of 18- to 20-week-old mice resulted in the formation of multiple hepatic metastases. Selective transduction of developing hepatic metastases was demonstrated using a beta-galactosidase-expressing recombinant adenovirus. Cytosine deaminase (CD) can metabolize 5-fluorocytosine into the chemotherapeutic reagent 5-fluorouracil (5FU). The antitumoral potential of this suicide gene therapy approach was explored by systemic application of a recombinant replication-deficient adenovirus encoding for the bacterial CD gene under the control of the cytomegalovirus promoter (Ad.CMV-CD). Injection into the tail vein of tumor-bearing mice resulted in delayed tumor growth with significant reduction in hepatic metastases. The potential of this experimental approach for possible future clinical applications was evaluated by investigating adenoviral transduction efficiency, 5FU sensitivity, and 5-fluorocytosine-dependent Ad.CMV-CD toxicity in a variety of human colon cancer cell lines. Although the murine cell lines MCA26 and CC36 were highly sensitive to 5FU, the human colon cancer cell lines showed a 1-100 times higher resistance to 5FU. Specific Ad.CMV-CD toxicity correlates with 5FU toxicity. Transduction efficiency in human colon carcinoma cell lines was shown to be 10-1700 times higher compared with murine cell lines, thus compensating for 5FU resistance. In conclusion, suicide gene therapy using CD may be promising as an adjuvant treatment regimen for hepatic micrometastases of human colon carcinoma.

A rapid and versatile method for harnessing scFv antibody fragments with various biological effector functions.

A versatile expression vector is described for the rapid construction and evaluation of bispecific scFvs and scFv-based fusion proteins. An important feature of this vector is the presence of two multiple cloning sites (MCS) separated by an in frame linker sequence. The first MCS was specifically designed to contain unique SfiI and NotI restriction enzyme sites that can be used for directional and in frame insertion of scFvs (or potentially any molecule) selected from established phage-display systems. Using this new vector, a functional bs-(scFv)(2) (2C11-MOC31) was constructed for retargeted T-cell cytotoxicity towards EGP2 positive tumor cells. The vector was also used for grafting of a number of promising biological effector principles onto scFv MOC31, including the prodrug converting enzyme cytosine deaminase, the anti-angiogenic factor angiostatin, and the thrombogenic molecule tissue factor. We aimed at producing biologically active fusion proteins by directing them through the endoplasmic reticulum-based protein folding machinery of eukaryotic cells (COS-7) using a kappa light chain leader, thereby taking advantage of the associated quality control mechanisms that allow only fully folded and processed fusion proteins to be secreted into the medium. Supernatants derived from fusion protein transfected COS-7 cells, which were transiently transfected at low transfection rates, were directly assayed for the biological and/or targeting activity of the excreted fusion proteins without any prior purification steps. This procedure might help to identify those fusion proteins that have favourable characteristics like stability and biological activity in the presence of serum and at low protein concentrations. Targeted delivery of all effector principles was subsequently assessed in an in vitro model system. The method we devised is both rapid and versatile and can be useful to construct and identify series of new chimeric proteins with enhanced therapeutic potential in human cancer therapy.

[Experimental study on human pancreatic carcinoma treated by cytosine deaminase gene therapy].

OBJECTIVE: To explore the possibility of Escherichia coli cytosine deaminase (CD) gene on human pancreatic carcinoma gene therapy. METHODS: Recombinant adenoviruses containing a carcinoembryonic antigen (CEA) promoter were transiently introduced into SW1990, Capan-2 and Hela cells, separately. The expression of CD gene mRNA was examined by RT-PCR. CD protein level in the transduced cells was analyzed by Western blotting. The sensitivity of the cells to 5-fluorocytosine (5-FC) was determined by MTT assay. RESULTS: A specific expression of cytosine deaminase gene by adenovirus-mediated transfer exhibited only in SW1990 cells (CEA-producing). Transduction of CD gene resulted in significant sensitivity of SW1990 cells to 5-FC. The anticancer effect was seen in vivo in SW1990 xenografts nude mice with in situ CD gene transduction. CONCLUSION: The targeted expression of CD gene combined prodrug 5-FC may be a potential approach for gene therapy for human pancreatic carcinoma.

Apoptosis induced by 5-flucytosine in human pancreatic cancer cells genetically modified to express cytosine deaminase.

AIM: To elucidate the pattern of 5-flucytosine (5-FC)-induced apoptosis and its role in gene therapy of human pancreatic cancer. METHODS: The human pancreatic cancer SW1990 cells (CEA-producing) were infected with recombinant adenoviruses (Adex1CEA-prCD or Adex1CEA-prZ). Expression of CD gene protein was examined by western blot. Apoptosis induced by 5-FC in human pancreatic cancer SW1990 cells genetically modified to express cytosine deaminase was observed by means of electron microscopy, DNA electrophoresis, and flow cytometry analysis techniques. RESULTS: The SW1990 cells infected with Adex1CEA-prCD were treated with 5-FC at 100 mumol.L-1 for 48 h, and cell apoptosis was observed. Typical apoptosis morphological feature appeared and DNA ladder could be demonstrated on DNA electrophoresis. Apoptosis peak was also showed by flow cytometry. Apoptotic cells accounted for 34.6% of the cell population. Cells in G1, S, and G2/M phase of cell cycle were 64%, 11%, and 7%, respectively. CONCLUSION: The apoptosis induced by 5-FC may be a primary mechanism in CD gene therapy of pancreatic cancer.

In vivo and in vitro glioma cell killing induced by an adenovirus expressing both cytosine deaminase and thymidine kinase and its association with interferon-alpha.

An adenovirus, AdCDTK, expressing both bacterial cytosine deaminase (CD) and herpes simplex virus thymidine kinase (HSVTK) was constructed and introduced into glioma cells. AdCDTK selectively rendered glioma cells sensitive to both 5-fluorocytosine (5-FCyt) and ganciclovir (GCV) (termed AdCDTK/5-FCyt-GCV). AdCDTK/5-FCyt-GCV not only potently mediated apoptosis and the arrest of glioma cell growth in vitro, but also significantly increased the survival time of glioma-bearing rats as compared with controls. The 90-day survival time was observed in 50% of rats. Interferon-alpha (IFN-alpha) further enhanced the tumor cell killing of AdCDTK/5-FCyt-GCV. In the group of AdCDTK/5-FCyt-GCV/IFN-alpha, the average survival time was significantly increased, and the average tumor size was smaller than that in the group of AdCDTK/5-FCyt-GCV. Ninety-day survival increased from 50% in the group of AdCDTK/5-FCyt-GCV to 75% in the group of AdCDTK/5-FCyt-GCV/IFN-alpha. Complete tumor regression was observed in 50% of rats in the group of AdCDTK/5-FCyt-GCV/IFN-alpha. The data indicate that AdCDTK/5-FCyt-GCV induces glioma cell killing greater than that induced by either CD/5-FCyt or HSVTK/GCV alone. IFN-alpha synergistically enhances this effect by increasing apoptosis.

Systemic administration of a recombinant vaccinia virus expressing the cytosine deaminase gene and subsequent treatment with 5-fluorocytosine leads to tumor-specific gene expression and prolongation of survival in mice.

Suicide gene therapy using the cytosine deaminase (CD) gene and 5-fluorocytosine (5-FC) has shown promising results for the treatment of colon carcinoma cells in vitro. Efficient viral infection and tumor-specific gene delivery is crucial for clinically measurable treatment effects. After proving efficient gene transfer in vitro, we demonstrate here that genes can be delivered to metastatic liver tumors in vivo in a highly selective manner using systemic delivery of a thymidine kinase-deleted (TK-) recombinant vaccinia virus (Western Reserve strain). When the vector was administered systemically in C57BL/6 mice or nude/athymic mice with established disseminated MC38 liver metastases, transgene expression in tumors was usually 1,000 to 10,000-fold higher compared with other organs (n = 160; P < 0.0001). This tumor-specific gene transfer leads to significant tumor responses and subsequent survival benefits after the transfer of the CD gene to liver metastases and subsequent systemic treatment with the prodrug 5-FC (P < 0.0001). We describe reporter gene and survival experiments both in immunocompetent and athymic nude mice, establishing a gene expression pattern over time and characterizing the treatment effects of the virus delivery/prodrug system. Cure rates of up to 30% in animals with established liver metastases show that suicide gene therapy using TK- vaccinia virus as a vector may be a promising system for the clinical application of tumor-directed gene therapy.

Inhibition of human immunodeficiency virus type 1 by Tat/Rev-regulated expression of cytosine deaminase, interferon alpha2, or diphtheria toxin compared with inhibition by transdominant Rev.

A retroviral vector was designed to express toxic proteins only in the presence of the HIV-1 Rev and/or Tat protein(s). The design of this vector incorporates an HIV-specific expression cassette that consists of three elements: the U3R region of the HIV-1 IIIB LTR provides the promoter and Tat-responsive element, a modified intron derived from the human c-src gene facilitates the splicing of inserted genes, and the HIV-1 RRE region enhances the transport of unspliced mRNAs. To further limit potential readthrough transcription, the expression cassette was inserted in the reverse transcriptional orientation relative to the retroviral vector LTR. Three different genes, interferon alpha2, diphtheria toxin (DT-A), and cytosine deaminase, were inserted into this vector. Tat and Rev inducibility was demonstrated directly by a >300-fold induction of interferon production and functionally by a decrease in colony-forming units when a Tat and Rev expression vector was titered on HeLa cells harboring the inducible DT-A cassette. The Tat-inducible cytosine deaminase gene was tested in the Sup-T1 T cell line and shown to inhibit HIV-1 production only when engineered cells were grown in the presence of 5-fluorocytosine. To test the ability of this system to inhibit HIV-1 infection in bulk PBL cultures, a series of transduction and challenge experiments was initiated with both the interferon and DT-A vectors. Protection against infection was documented against three HIV strains in PBLs. Last, the interferon and DT-A vectors were compared with a vector encoding a transdominant Rev protein and were shown to mediate equal or greater inhibition of HIV-1.

Efficacy of adenovirus-mediated CD/5-FC and HSV-1 thymidine kinase/ganciclovir suicide gene therapies concomitant with p53 gene therapy.

Recent evidence has suggested that tumor cells having a wild-type p53 status are more sensitive to chemotherapeutic agents and radiation than cells that lack functional p53. The heightened sensitivity of wild-type p53 cells is thought to be attributable to their propensity to undergo p53-mediated apoptosis after insult. Given that suicide gene therapy is essentially tumor-targeted chemotherapy, we examined the hypothesis that coexpression of wild-type p53 could enhance the efficacy of adenovirus-mediated suicide gene therapy. Human Hep3B and SK-OV-3 cells, which are null for p53, were infected with a pair of replication-deficient adenoviruses that expressed a cytosine deaminase/herpes simplex virus thymidine kinase (CD/HSV-1 TK) fusion gene without (fusion gene nonreplicative adenovirus, FGNR) or with (FGNRp53) the wild-type human p53 gene. The sensitivity of cells to the CD/5-fluorocytosine (CD/5-FC) and HSV-1 TK/ ganciclovir (GCV) enzyme/prodrug systems was determined in vitro and in vivo. Coexpression of p53 did not enhance the cytotoxicity of either the CD/5-FC or HSV-1 TK/GCV system in vitro. The failure to observe an effect of p53 could not be explained on the basis of insufficient or transient p53 expression, because FGNRp53-infected cells growth arrested in G1, induced Bax, and underwent apoptosis at an increased rate after prodrug treatment, particularly when the adenovirus E1A protein was present. Intratumoral injection of FGNRp53 concomitant with single or double pro-drug therapy resulted in a tumor growth delay that was equal to or less than that observed with the FGNR virus. Our results indicate that coexpression of p53 may not necessarily improve the efficacy of adenovirus-mediated CD/ 5-FC and HSV-1 TK/GCV suicide gene therapies in vivo.