Cytosine Deaminase Gene Research Articles

The potential therapeutic gain of radiation-associated gene therapy with the suicide gene cytosine deaminase

Purpose : To estimate the concentration of 5-fluorocytosine (5-FC), necessary for conversion to 5-fluorouracil (5-FU) in tumours transduced with the gene cytosine deaminase (CD), to achieve clinically significant radiosensitization to radiotherapy. Materials and methods : Starting with a tumour control probability (TCP) of 37% from radiotherapy of 66 Gy in 2 Gy fractions, estimates were made of increase in TCP expected from sensitizer enhancement ratios (SER) of 1.1, 1.2, etc. SER values for 5-FU were obtained from a literature review. Clinical toxicity of 5-FC is also reviewed. Results : 5-FU has been reported to be an effective radiosensitizer if maintained for several days after each irradiation at concentrations of 0.6-0.9 mug/ml in surrounding medium. 5-FC is well tolerated by patients at concentrations of 25-100mug/ml (average 60mug/ml) for 6 weeks in standard antifungal treatment. Sufficient 5-FU should be available if conversion efficiency from 5-FC is 1-3%. SER values of 1.1 to 1.2 should be achievable with daily 2 Gy fractions. In vitro and xenograft experiments are reviewed and they do not contradict the conclusions. Conclusions : Increases in tumour control of 20 to 40% can be expected, which should be detectable in a 2-arm randomized trial of 260 (for 20%) or 60 (for 40%) patients.

Cytosine deaminase suicide gene therapy for peritoneal carcinomatosis.

Gene therapy is a novel therapeutic approach that might soon improve the prognosis of some cancers. We investigated the feasibility of cytosine deaminase (CD) suicide gene therapy in a model of peritoneal carcinomatosis. DHD/K12 colorectal adenocarcinoma cells transfected in vitro with the CD gene were highly sensitive to 5-fluorocytosine (5-FC), and a bystander effect could also be observed. Treating CD+ cells with 5-FC resulted in apoptosis as detected by terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate nick-end labeling. In vitro, several human cell lines derived from ovarian or colorectal carcinomas, as well as the rat glioblastoma 9 L cell line, responded to CD/5-FC and showed a very strong bystander effect. 5-FC treatment of peritoneal carcinomatosis generated in syngeneic BDIX rats by CD-expressing DHD/K12 cells led to a complete and prolonged response and to prolonged survival. Our study thus demonstrated the efficacy of CD suicide gene therapy for the treatment of peritoneal carcinomatosis.

The nitroreductase/CB1954 combination in Epstein-Barr virus-positive B-cell lines: induction of bystander killing in vitro and in vivo.

Epstein-Barr virus (EBV)-based gene delivery vectors that preferentially express toxic genes in EBV-infected cells could be used to target EBV-positive tumors for destruction. We have shown previously that the cytosine deaminase (CD) enzyme, which converts the prodrug 5-fluorocytosine (5-FC) into the toxic compound 5-fluorouracil efficiently kills EBV-positive cells in the presence of 5-FC, with a substantial bystander killing effect in vitro and in vivo. To identify the optimal enzyme/prodrug combination for treating EBV-positive lymphomas, we have compared the effectiveness of the CD/5-FC combination with the nitroreductase (NTR)/CB1954 combination for killing EBV-positive B-cell lines. NTR metabolizes CB1954 into an alkylating agent that cross-links DNA. When the CD gene or the NTR gene were transfected into two different EBV-positive B-cell lines in vitro, approximately 90% of cells were killed in a prodrug-dependent manner, although the transfection efficiency was <5%. However, severe combined immunodeficient mouse tumors containing either 30% or 100% of NTR-expressing Burkitt lymphoma (Jijoye) cells were growth inhibited, but not cured, by treatment with intraperitoneal CB1954 (20 mg/kg/day) for 10 days. These results suggest that the NTR/CB1954 combination induces efficient bystander killing of EBV-positive B-cell lines in vitro but may not be as effective as the CD/5-FC combination for treating B-cell lymphomas in vivo.

In vivo efficacy and toxicity of 5-fluorocytosine/cytosine deaminase gene therapy for malignant gliomas mediated by adenovirus.

We evaluated the therapeutic efficacy and neurotoxicity of adenovirus-mediated transduction of the cytosine deaminase (CD) gene and 5-fluorocytosine (5-FC) for experimental malignant brain tumors. The 5-FC sensitivity in 9 L cells infected by an adenovirus vector expressing CD (AdexCACD) was increased 1700-fold compared with control cells. Rats bearing 9 L brain tumors were treated with an intratumoral injection of AdexCACD followed by intraperitoneal administration of 5-FC. The rats demonstrated remarkable inhibition of tumor growth by magnetic resonance imaging, and 7 of 10 rats survived for >90 days. To evaluate the potential side-effects of the 5-FC/CD gene therapy, rats were treated with an intracerebral injection of AdexCACD into the right basal ganglia and with 5-FC. The magnetic resonance imaging showed a highly enhanced area on the gadollinium-enhanced T1-weighted image at 18 days postinjection. Pathologically, this corresponded to an area of necrosis with surrounding apoptotic cells. In addition, there was demyelination and gliosis with enlargement of the lateral ventricles. These results suggest that the 5-FC/CD gene therapy may provide an anticancer effect for malignant brain tumors in humans, but also show that there are neurotoxic effects on normal brain tissue.

Cytosine deaminase/5-fluorocytosine-based vaccination against liver tumors: evidence of distant bystander effect.

The cytosine deaminase gene of Escherichia coli converts the nontoxic compound 5-fluorocytosine into 5-fluorouracil (5-FU), thereby acting as a suicide gene when introduced into cancer cells, killing the cells when they are exposed to 5-fluorocytosine. We analyzed the efficacy of using cytosine deaminase-bearing cancer cells as an autologous tumor vaccine in a rat model that mimics liver metastasis from colon carcinoma. We introduced a plasmid vector containing the E. coli cytosine deaminase gene into a BDIX rat colon carcinoma cell line. Intrahepatic injection of the modified cells in syngeneic animals generates a single experimental liver "suicide tumor." We then analyzed the effect of 5-fluorocytosine treatment in terms of regression of cytosine deaminase-expressing cells in vivo as well as protection against wild-type cancer cells. Treatment with 5-fluorocytosine induced regression of cytosine deaminase-expressing (CD+) tumors, with seven of 11 treated animals being tumor free at the end of 30 days and a statistically significant difference in tumor volumes between treated and control animals (two-sided P<.0001). Intrahepatic injection of CD+ cells followed by 5-fluorocytosine treatment rendered the treated animals resistant to challenge with wild-type tumor cells, with no (zero of seven) treated animals developing wild-type tumors in contrast to all (four of four) control animals. Moreover, in animals with established wild-type liver tumors, injection of CD+ tumor cells followed by 5-fluorocytosine treatment produced a statistically significant increase in survival time (two-sided P<.0001). In vivo immunodepletion and immunohistologic analysis of experimental tumors indicate that natural killer cells are the major immune component involved in this antitumor effect. Taken together, these results suggest the potential use of suicide gene-modified tumor cells as therapeutic vaccines against liver metastasis from colon carcinoma.

Analysis of the human carcinoembryonic antigen promoter core region in colorectal carcinoma-selective cytosine deaminase gene therapy.

We isolated a 204-base pair carcinoembryonic antigen (CEA) promoter core region from a CEA-producing human colorectal carcinoma (CRC) and constructed retrovirus vectors carrying the expression cassette consisting of the CEA promoter core region and the cytosine deaminase (CD) gene. pCD2 retrovirus carrying the CD gene directed by the retrovirus long terminal repeat promoter served as a control vector. An in vitro study showed that the CEA promoter conferred CEA-producing cell-selective CD expression, specifically when the CD expression cassette was inserted into the 3' long terminal repeat of the retrovirus vector. CD-modified CRC xenografts in nude mice were sensitive to 5-fluorocytosine and caused a profound bystander effect on the unmodified CRC. When nude mice harboring intraperitoneally disseminated CRCs were injected intraperitoneally with the CD expression cassette-carrying retrovirus-producing cells, CD transduction into the disseminated CRCs and bone marrow (BM) was observed. CD expression was, however, restricted to CRCs, and it was observed in both CRCs and BM of mice injected with pCD2 retrovirus-producing cells, resulting in better therapeutic outcomes without BM suppression. These results indicate that effective and safe in vivo gene therapy for CRC may be feasible by transferring the CD gene controlled by the CEA promoter core region.

CYTOTOXICITY OF ADENOVIRAL-MEDIATED CYTOSINE DEAMINASE PLUS 5-FLUOROCYTOSINE GENE THERAPY IS SUPERIOR TO THYMIDINE KINASE PLUS ACYCLOVIR IN A HUMAN RENAL CELL CARCINOMA MODEL

Purpose An estimated 11,600 Americans will die of renal cell carcinoma in 1998. The lack of effective chemotherapy or radiotherapy requires the investigation of novel treatment modalities. We compared two forms of toxic gene therapy, cytosine deaminase (CD) plus 5-fluorocytosine (5-FC) and thymidine kinase (TK) plus acyclovir (ACV), in pre-clinical models of human renal cell carcinoma. Materials and Methods Replication-deficient recombinant adenoviral vectors containing the Rous sarcoma virus promoter driving CD (Ad-RSV-CD) or TK (Ad-RSV-TK) gene expression were constructed and tested for in vitro cell-killing assays at various viral multiplicity of infection (MOI) and in vivo for growth inhibition of a human renal cell carcinoma, SK-RC-29 models. Subcutaneous tumors of SK-RC-29 were examined by electron microscopy for presence of intercellular gap junctions. Levels of expression of the gap junctional associated connexin 43 protein in SK-RC-29, 31, 38, 42, 52 human RCC cell lines were examined by western immunoblotting. Results In vitro cell-killing assay comparing Ad-RSV-CD/5F-C and Ad-RSV-TK/ACV at a wide range of MOI (2.5 to 20) revealed superior cell-kill by Ad-RSV-CD/5-FC over Ad-RSV-TK/ACV. Consistent with these results, we observed that Ad-RSV-CD/5-FC but not Ad-RSV-TK/ACV demonstrated a significant in vivo tumor growth inhibition. These results are corroborated by the lack of gap junctions in SK-RC-29 subcutaneous tumors by the electron microscopy and the absence of connexin-43 in all five human RCC cell lines by western immunoblotting. Conclusion We have demonstrated in this study that Ad-RSV-CD/5-FC is superior to Ad-RSV-TK/ACV for the treatment of human RCC in cell culture and animal models. The results are supported by the lack of gap junctional communication between RCC cells assessed by connexin-43 expression.

In vivo gene transfer of a suicide gene under the transcriptional control of the carcinoembryonic antigen promoter results in bone marrow transduction but can avoid bone marrow suppression.

We constructed the CEA419/CD retrovirus vector carrying the cytosine deaminase (CD) gene directed by the carcinoembryonic antigen (CEA) promoter. pCD2 retrovirus vector carrying the CD gene directed by the retrovirus long terminal repeat promoter was also used. When mice bearing intraperitoneally disseminated colorectal carcinomas (CRCs) were infused intraperitoneally with pCD2 or CEA419/CD retrovirus-producing cells, a CD fragment was detected in CRCs and bone marrow cells. It was shown that the CD gene was expressed both in CRCs and in the bone marrow of animals infused with pCD2 retrovirus-producing cells, while the CD gene was expressed solely in CRCs of animals infused with CEA419/CD retrovirus-producing cells. These results indicate that the use of a tumor-selective promoter may warrant the safety of in vivo gene therapy using suicide genes.

Genetic Prodrug Activation Therapy for Breast Cancer: A Phase I Clinical Trial oferbB-2–Directed Suicide Gene Expression

This trial was designed to test the safety and efficacy of a tumor-specific genetic prodrug activation therapy targeted by use of the human erbB-2 gene promoter. The erbB-2 oncogene is overexpressed in approximately 20% of cases of breast cancer and is associated with poor prognosis. Twelve breast cancer patients received transcriptionally targeted gene therapy in a phase I clinical trial using direct intratumoral injection of plasmid construct combined with systemic administration of prodrug. The genetic prodrug activation therapy is specifically targeted to erbB-2-overexpressing breast cancer cells by use of a therapeutic cassette that contains the Escherichia coli cytosine deaminase gene driven by the tumor-specific erbB-2 promoter, thus allowing activation of fluorocytosine to the active cytotoxic fluorouracil only within tumor cells that express the oncogene. The approach was shown to be safe and to result in targeted gene expression in up to 90% of cases. Using a number of different assays, we demonstrated that significant levels of expression of the suicide gene were specifically restricted to erbB-2-positive tumor cells, confirming the selectivity of the approach. The results of this study, the first targeted gene therapy for breast cancer and the first to use the cytosine deaminase system in human subjects, are encouraging for the development of genetic prodrug activation therapies that exploit the transcriptional profile of cancer cells.

Cytosine deaminase/5-fluorocytosine gene therapy can induce efficient anti-tumor effects and protective immunity in immunocompetent mice but not in athymic nude mice.

Murine hepatocellular carcinoma cells were retrovirally transduced with the bacterial cytosine deaminase (CD) gene. CD-transduced cells exhibited more than 120-fold higher sensitivity to 5-fluorocytosine (5-FC) compared with parental cells. When syngeneic immunocompetent mice were inoculated s.c. with parental hepatocellular carcinoma cells containing as little as 5% CD-transduced cells, significant inhibition of tumor formation was induced by 5-FC treatment. Furthermore, established solid tumors in immunocompetent mice containing only 5% CD-transduced cells were infiltrated markedly with CD4- and CD8+ T lymphocytes and macrophages by 5-FC treatment, such that significant reduction or even complete regression of tumors was observed. These tumor-free mice resisted subsequent rechallenge with wild-type tumor. Conversely, when athymic nude mice were inoculated with a cell mixture containing CD-transduced cells and parental cells at a ratio of 40:60, all developed tumors despite 5-FC treatment. Our results indicate that gene therapy using the CD/5-FC system can induce efficient anti-tumor effects and protective immunity in immunocompetent mice but not in athymic immunodeficient mice, suggesting that the host's immunocompetence may be a critical factor for achieving successful gene therapy against cancer.

Ex vivo delivery of suicide genes into melanoma cells using epidermal growth factor receptor-specific Fab immunogene.

The Fab fragment of monoclonal antibody B4G7 against human epidermal growth factor (EGF) receptor was conjugated with cationic poly‐l‐lysine and the resulting conjugate was further complexed with reporter genes or therapeutic genes. This Fab/DNA complex was designated as “Fab immunogene”. The Fab immunogene transfer in vitro was mediated through the EGF receptors in two melanoma cell lines. The frequency of cells expressing β‐galactosidase (β‐Gal) reporter gene was approximately 1%. The induction of suicide effects after Fab immunogene transfer of herpes simplex virus thymidine kinase (TK) or Escherichia coli cytosine deaminase (CD) gene was quite remarkable, and the growth of melanoma cells was inhibited for over 7 days in the presence of ganciclovir (GCV) or 5‐fluorocytosine (5‐FC). Similarly, when melanoma cells treated in vitro with the Fab immunogene carrying TK or CD were transplanted into the back of nude mouse, subsequent systemic administration of GCV or 5‐FC effectively suppressed the growth of tumors, indicating the occurrence of in vivo suicide effects.

Negative selection driven by cytosine deaminase gene in Lycopersicon esculentum hairy roots

The bacterial cytosine deaminase ( cd) gene was employed as a negative selection marker in transgenic tomato ( Lycopersicon esculentum var. cerasiforme) hairy roots. The cd gene was introduced in tomato cells using a modified binary vector in the Agrobacterium rhizogenes wild type strain 8196. The transconjugant strain was obtained through triparental mating and then inoculated on tomato stem fragments. The roots that appeared at the inoculation site were isolated and subcultured until axenic root clones were established. These roots were tested for their ability to grow in medium containing 5-fluorocytosine (5-FC), which becomes toxic in the presence of cytosine deaminase. The data, together with molecular analysis of the CD-T-DNA transferred, clearly show that expression of cd in root cells provides a strong negative selection, completely inhibiting root growth. This negative selection was obtained in roots where only one copy of the cd gene was integrated in the cell genome, providing a potential tool for mutagenesis and transposon mobilization studies.

Effective and safe gene therapy for colorectal carcinoma using the cytosine deaminase gene directed by the carcinoembryonic antigen promoter.

We have recently isolated carcinoembryonic antigen (CEA) promoter regions consisting of 419 bp and 204 bp from CEA-producing human colorectal carcinoma (CRC). We constructed CEA419/CD and CEA204/CD retroviruses carrying the bacterial cytosine deaminase (CD) gene directed by the CEA promoter regions. pCD2 retroviruses carrying the CD gene directed by the retrovirus long terminal repeat promoter were also used. CEA419/CD or CEA204/CD retrovirus-infected CRC cells were found to be susceptible to 5-fluorocytosine (5-FC), while non-CRC cells infected with the same retroviruses were not. CD-transduced CRC xenografts in nude mice were sensitive to 5-FC treatment, resulting in arrest of tumor growth. When mice with intraperitoneally disseminated CRCs were given intraperitoneal injections of CEA419/CD retrovirus-producing cells followed by 5-FC treatment, significantly prolonged survival rates were observed compared with animals injected with pCD2 retrovirus-producing cells followed by 5-FC treatment. Importantly, bone marrow suppression was not observed in animals injected with CEA419/CD retrovirus-producing cells and 5-FC, while profound bone marrow suppression was observed in those injected with pCD2 retrovirus-producing cells and 5-FC. These results indicate that effective and safe in vivo gene therapy for advanced CRC may be feasible by transferring the CD gene controlled by the CEA promoter followed by 5-FC treatment.

Comparison of Gene Therapy with the Herpes Simplex Virus Thymidine Kinase Gene and the Bacterial Cytosine Deaminase Gene for the Treatment of Hepatocellular Carcinoma

Background: Bystander effects induced by suicide gene/prodrug systems play an essential role in achieving successful antitumor effects. Although it has been shown in several in vitro studies that the bacterial cytosine deaminase (CD) gene/5-fluorocytosine (5-FC) system is superior to the herpes simplex virus thymidine kinase (HSV-TK) gene/ganciclovir (GCV) system, we examined here which suicide gene system was more promising in vivo for the treatment of hepatocellular carcinoma (HCC). Methods: BNL1ME A.7R.1 murine HCC cells were retrovirally transduced with the HSV-TK or CD gene, and bystander effects caused by the appropriate prodrug treatment were examined not only in vitro but also in vivo. Results: The CD/5-FC system was superior to the HSV-TK/GCV system in HCC cell elimination in vitro. The bystander effect of the HSV-TK/GCV was shown to be substantially dependent on cell-to-cell contact, whereas that of the CD/5-FC was not. However, antitumor effects on HCC and tumor immunity to parental HCC induced by the HSV-TK/GCV system were not inferior and even superior to those induced by the CD/5-FC system. Bystander effects induced by the suicide gene/prodrug systems in immunocompetent syngeneic mice were much more profound than those induced in vitro. However, significant bystander effects were not observed in athymic nude mice. Conclusions: These results suggest that both HSV-TK/GCV and CD/5-FC systems are useful for the treatment of HCC. The results also suggest that T-cell-mediated immune responses elicited by the suicide gene/prodrug systems play a substantial role in antitumor effects in vivo.

Stable transformation of Toxoplasma gondii based on a pyrimethamine resistant trifunctional dihydrofolate reductase-cytosine deaminase-thymidylate synthase gene that confers sensitivity to 5-fluorocytosine

To improve genetic models available for the analysis of apicomplexan protozoan parasites, bacterial sequences encoding the 427 amino acid cytosine deaminase (CD) gene were fused, in-frame, to an engineered linker domain of the high level pyrimethamine resistant form of the parasite bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) gene. Toxoplasma gondii was transformed with the plasmid containing the fused pyrimethamine resistant dihydrofolate reductase-cytosine deaminase-thymidylate synthase (DHFRm2m3-CD-TS) gene and parasites were selected in a high level of pyrimethamine. Transfected parasites that acquired resistance to pyrimethamine were cloned and evaluated for expression of the CD genetic marker. CD transgenic parasites acquired a high sensitivity to 5-fluorocytosine due to the intraparasitic conversion of this non-toxic prodrug to the cytotoxic compound 5-fluorouracil. Exogenously supplied cytosine or uracil rescued the growth of CD transgenic T. gondii parasites that were cultured in the presence of cytotoxic concentrations of 5-fluorouracil or 5-fluorocytosine. Bacterial CD fused to the pyrimethamine resistant DHFR-TS marker provides a novel genetic tool for new positive and negative genetic selection strategies in several protozoan parasites. An advantage of the CD genetic marker is that it is derived from a bacterial gene and can therefore be used in nearly any parasite genetic background for negative selection. This novel system should facilitate new approaches for the development of improved model genetic systems for the biological investigation of apicomplexan parasites.

Selectable marker-free transgenic plants without sexual crossing: transient expression of cre recombinase and use of a conditional lethal dominant gene.

Transgenic tobacco plants were produced that contained single-copy pART54 T-DNA, with a 35S-uidA gene linked to loxP-flanked kanamycin resistance (nptII) and cytosine deaminase (codA) genes. Retransformation of these plants with pCre1 (containing 35S transcribed cre recombinase and hygromycin (hpt) resistance genes) resulted in excision of the loxP-flanked genes from the genome. Phenotypes of progeny from selfed-retransformed plants confirmed nptII and codA excision and integration of the cre-linked hpt gene. To avoid integration of the hpt gene, and thereby generate plants totally free of marker genes, we attempted to transiently express the cre recombinase. Agrobacterium tumefaciens (pCre1) was cocultivated with leaf discs of two pART54-transformed lines and shoots were regenerated in the absence of hygromycin selection. Nineteen of 773 (0.25%) shoots showed tolerance to 5-fluorocytosine (5-fc) which is converted to the toxic 5-fluorouracil by cytosine deaminase. 5-fc tolerance in six shoots was found to be due to excision of the loxP-flanked region of the pART54 T-DNA. In four of these shoots excision could be attributed to cre expression from integrated pCre1 T-DNA, whereas in two shoots excision appeared to be a consequence of transient cre expression from pCre1 T-DNA molecules which had been transferred to the plant cells but not integrated into the genome. The absence of selectable marker genes was confirmed by the phenotype of the T1 progeny. Therefore, through transient cre expression, marker-free transgenic plants were produced without sexual crossing. This approach could be applicable to the elimination of marker genes from transgenic crops which must be vegetatively propagated to maintain their elite genotype.

Provision of positive and negative selections in retroviral vectors containing the cytosine deaminase gene.

The E. coli cytosine deaminase (CD) provides a negative selection system for suicide gene therapy as CD transfectants are eliminated following 5-fluorocytosine (5FC) treatment. Here we report a positive selection system for the CD gene using 5-fluorouracil (5FU) and cytosine in selection medium to screen for CD-positive transfectants. It is based on the relief of 5FU toxicity by uracil which is converted from cytosine via CD catalysis, as uracil competes with the toxic 5FU in subsequent pyrimidine metabolism. Hence, a retroviral vector containing the CD gene may provide both positive and negative selections after gene transfer. The CD transfectants selected with the positive selection system showed susceptibility to 5FC in subsequent negative selection in vitro and in vivo. Therefore, this dual selection system is useful not only for combination therapy with transgene and CD gene, but can also act to eliminate selectively transduced cells after the transgene has furnished its effects or upon undesired conditions if 5FC is applied for negative selection in vivo.

Ex vivo breast cancer cell purging by adenovirus-mediated cytosine deaminase gene transfer and short-term incubation with 5-fluorocytosine completely prevents tumor growth after transplantation.

Peripheral blood progenitor harvests of breast cancer patients are contaminated with tumor cells, suggesting a potential role for these cells in the relapse after high-dose chemotherapy. Whereas physical purging methods do not eliminate contaminating tumor cells completely, pharmacological purging, although highly efficient, is hampered by a strong nonspecific toxicity toward hematopoietic progenitor cells. Taking advantage of the high efficiency of adenovirus-mediated gene transfer to epithelial cells, we selectively loaded breast cancer cells in vitro with a cytotoxic drug by gene transfer of the prodrug-converting enzyme cytosine deaminase (AdCMV.CD) and 5-fluorocytosine (5-FC). Despite the low dose of vector administered, limited exposure to 5-FC, and transplantation only of viable tumor cells into SCID mice, all animals that received cells treated in vitro with AdCMV.CD plus 5-FC were completely free of tumor development. These data show that the selective loading of tumor cells with AdCMV.CD/5-FC might be useful for purging of autografts.

Adenovirus-mediated GM-CSF gene and cytosine deaminase gene transfer followed by 5-fluorocytosine administration elicit more potent antitumor response in tumor-bearing mice.

Antitumor effects of combined transfer of suicide and cytokine genes were investigated in this study. Adenovirus harboring E. coli cytosine deaminase gene (AdCD) and adenovirus harboring murine granulocyte-macrophage colony-stimulating factor gene (AdGMCSF) were used simultaneously for in vivo gene transfer in melanoma-bearing mice. Growth inhibition of established tumors and prolongation of survival period were observed more significantly in tumor-bearing mice after transfection with AdGMCSF and AdCD followed by continuous injection of prodrug 5-fluorocytosine (5FC) when compared with mice treated with control adenovirus AdlacZ/5FC, AdCD/5FC or AdGMCSF alone (P < 0.01). After combined therapy the expression of MHC-I (H-2Db) and B7-1 molecules on freshly isolated tumor cells increased greatly and more dendritic cells and CD8+ T cells infiltrated into the tumor mass. The activity of specific cytotoxic T lymphocytes was also found to be induced more significantly after the combined therapy. Further experiments showed that apoptosis of tumor cells and induction of antitumor immune response might be involved in the mechanisms of the tumor cell killing by the combined therapy. Our results demonstrated that combined transfer of the GM-CSF and CD suicide genes, being able to inhibit the growth of melanoma synergistically and induce specific antitumor immune response efficiently, thus addressing the drawbacks of suicide gene therapy or cytokine gene therapy which were proved to be not satisfactory when used alone, might be of therapeutic potential for gene therapy of cancer.

Cytosine Deaminase Adenoviral Vector and 5-Fluorocytosine Selectively Reduce Breast Cancer Cells 1 Million-Fold When They Contaminate Hematopoietic Cells: A Potential Purging Method for Autologous Transplantation

AbstractAd.CMV-CD is a replication incompetent adenoviral vector carrying a cytomegalovirus (CMV)-driven transcription unit of the cytosine deaminase (CD) gene. The CD transcription unit in this vector catalyzes the deamination of the nontoxic pro-drug, 5-fluorocytosine (5-FC), thus converting it to the cytotoxic drug 5-fluorouracil (5-FU). This adenoviral vector prodrug activation system has been proposed for use in selectively sensitizing breast cancer cells, which may contaminate collections of autologous stem cells products from breast cancer patients, to the toxic effects of 5-FC, without damaging the reconstitutive capability of the normal hematopoietic cells. This system could conceivably kill even the nondividing breast cancer cells, because the levels of 5-FU generated by this system are 10 to 30 times that associated with systemic administration of 5-FU. The incorporation of 5-FU into mRNA at these high levels is sufficient to disrupt mRNA processing and protein synthesis so that even nondividing cells die of protein starvation. To test if the CD adenoviral vector sensitizes breast cancer cells to 5-FC, we exposed primary explants of normal human mammary epithelial cells (HMECs) and the established breast cancer cell (BCC) lines MCF-7 and MDA-MB-453 to the Ad.CMV-CD for 90 minutes. This produced a 100-fold sensitization of these epithelial cells to the effects of 48 hours of exposure to 5-FC. We next tested the selectivity of this system for BCC. When peripheral blood mononuclear cells (PBMCs), collected from cancer patients during the recovery phase from conventional dose chemotherapy-induced myelosuppression, were exposed to the Ad.CMV-CD for 90 minutes in serum-free conditions, little or no detectable conversion of 5-FC into 5-FU was seen even after 48 hours of exposure to high doses of 5-FC. In contrast, 70% of 5-FC was converted into the cytotoxic agent 5-FU when MCF-7 breast cancer cells (BCCs) were exposed to the same Ad.CMV-CD vector followed by 5-FC for 48 hours. All of the BCC lines tested were shown to be sensitive to infection by adenoviral vectors when exposed to a recombinant adenoviral vector containing the reporter gene betagalactosidase (Ad.CMV-βgal). In contrast, less than 1% of the CD34-selected cells and their more immature subsets, such as the CD34+CD38− or CD34+CD33− subpopulations, were positive for infection by the Ad.CMV-βgal vector, as judged by fluorescence-activated cell sorting (FACS) analysis, when exposed to the adenoviral vector under conditions that did not commit the early hematopoietic precursor cells to maturation. When artificial mixtures of hematopoietic cells and BCCs were exposed for 90 minutes to the Ad.CMV-CD vector and to 5-FC for 10 days or more, a greater than 1 million fold reduction in the number of BCCs, as measured by colony-limiting dilution assays, was observed. To test if the conditions were damaging for the hematopoietic reconstituting cells, marrow cells collected from 5-FU–treated male donor mice were incubated with the cytosine deaminase adenoviral vector and then exposed to 5-FC either for 4 days in vitro before transplantation or for 14 days immediately after transplantation in vivo. There was no significant decrease in the reconstituting capability of the male marrow cells, as measured by their persistence in female irradiated recipients for up to 6 months after transplantation. These observations suggest that adenovirus-mediated gene transfer of the Escherichia coli cytosine deaminase gene followed by exposure to the nontoxic pro-drug 5-FC may be a potential strategy to selectively reduce the level of contaminating BCCs in collections of hematopoietic cells used for autografts in breast cancer patients.