Affiliations of author: Metabolism Branch, Division of Clinical Sciences, National Cancer Institute, and Clinical Gene Therapy Branch, National Human Genome Research Institute, Bethesda, MD.
Correspondence to: John C. Morris, M.D., National Institutes of Health, Bldg. 10, Rm. 4N115, Bethesda, MD 20892-1374 (e-mail: jmorris{at}mail.nih.gov).
A major focus of gene therapy for cancer has been the effort to introduce into cancer cells a number of foreign genes that encode enzymes that will selectively convert nontoxic prodrugs into toxic compounds, producing high local concentrations that result in tumor cell killingso-called "suicide" gene therapy. A number of enzyme/prodrug systems have been described [reviewed in (1)], including herpes simplex virus-thymidine kinase (HSV-tk)/ganciclovir (GCV) and Escherichia coli cytosine deaminase (CD)/5-fluorocytosine. A surprising early observation in many of these systems was that not every cell in a tumor need express the transgene to achieve meaningful cell killing and tumor regression (2,3). This phenomenon, the bystander effect, is defined as the ability of the genetically modified cells, in the presence of the prodrug, to cause cytotoxic effects in cells that lack the suicide gene. The result is that the fraction of cells killed is in excess of the fraction in the tumor that actually expresses the suicide gene (4). The bystander effect is a powerful enhancement of many suicide gene/prodrug systems that compensates for the inability of current vector systems to transduce all but a small fraction cells in a given tumor (5).
The degree and mechanism of the bystander effect differ with various cell lines and the enzyme/prodrug system studied. At the cellular level, several explanations have been advanced for the bystander effect; however, current evidence supports direct transfer of activated prodrug from the transgene-expressing cells to untransduced wild-type cells (6-8). In the HSV-tk/GCV system, in which the activated prodrug is highly ionized and is unable to diffuse across cell membranes, the bystander effect appears to be mediated by transfer of phoshorylated GCV through cellular gap junctions (6,8,9). Nonadherent cells and those with few gap junctions do not exhibit a significant HSV-tk/GCV bystander effect. 5-Fluorouracil (5-FU), the activated prodrug of the CD/5-fluorocytosine system, is a small nonpolar molecule that passively diffuses across the cell membrane down its concentration gradient, from CD+ cells to untransduced cells (7,10). The clinical importance of the bystander effect is illustrated by an early trial that involved transfer of the HSV-tk gene by stereotactic implantation of murine retroviral vector producer cells into patients with refractory brain tumors who were subsequently treated with GCV (11). Despite an estimated transduction efficiency of under 5%, responses were seen in five of 19 treated lesions.
Even with the ability to achieve local responses, any clinically useful gene therapy must address the fact that most cancer deaths are the result of uncontrolled metastatic disease. There is currently no gene delivery system that can approach the transduction efficiency and selectivity required for successful systemic treatment of metastatic cancer. These issues have prompted investigation of the potential vaccine effect of enzyme/prodrug therapy. Killing of cancer cells by coexpression of foreign bacterial or viral proteins may lead to enhancement of the immune response to normally weak tumor antigens (12,13). Several groups (14-16) have reported the development of resistance to challenge with wild-type cells from the same tumor cell line in animals that had been successfully treated with tumor cells transduced with either the HSV-tk or the CD gene for enzyme/prodrug therapy. Researchers (17,18) have also described regression of wild-type tumors implanted in a remote site within an animal after GCV treatment of HSV-tk-transduced tumors. This phenomenon has been termed the "bystander effect at a distance." Histologic examination of the regressing tumors shows inflammatory infiltrates, predominantly composed of CD8+ T cells and macrophages (14,19). These findings have been confirmed in animals bearing CD-expressing tumors that have been treated with 5-fluorocytosine (15). Treatment of HSV-tk-expressing tumors with GCV has been shown to increase the expression of major histocompatibility complex class I molecules, the costimulatory surface molecule B7, which is the ligand for the CD28 receptor on T cells, and surface intercellular adhesion molecules (16,20) as well as to induce development of a tumor-specific cytotoxic T-lymphocyte response (20).
In this issue of the Journal, Pierrefite-Carle et al. (21) address the anticancer vaccine effect of the CD/5-fluorocytosine enzyme/prodrug system. CD deaminates the nontoxic antifungal drug 5-fluorocytosine to generate the antitumor agent 5-FU (22). Cells that express CD are sensitized, by 500-fold to 2000-fold, to 5-fluorocytosine (3,23). Treatment with 5-fluorocytosine causes regression of CD-expressing tumors and has been shown to have a strong local bystander effect in many cell lines, with the presence of as little as 2% CD+ cells in a tumor resulting in cure of the animals (3,7,23,24). The CD/5-fluorocytosine system is currently undergoing clinical evaluation (25).
Pierrefite-Carle et al. (21) generated CD+ rat DHD/K12/PROb (PROb) colon cancer cells by plasmid transfection and selection. Not surprisingly, tumor cells that expressed CD (PRObCD+ cells) and that were injected into the liver of rats failed to form tumors after the rats were treated with 5-fluorocytosine beginning 24 hours after inoculation. Wild-type PROb cells readily formed measurable tumors despite 5-fluorocytosine treatment. None of seven animals given an injection of wild-type PROb cells in the opposite lobe of the liver of rats receiving 5-fluorocytosine treatment for previously implanted PRObCD+ tumors developed tumors, whereas all of the control animals developed measurable tumors. Rats that developed wild-type PROb liver tumors and that were inoculated with PRObCD+ cells in the opposite lobe of the liver and then treated with 5-fluorocytosine had an improved survival when compared with the survival among control rats (median survival, 154 days versus 91 days; P<.0001). These results are suggestive of a vaccine effect against wild-type PROb cells stimulated by the 5-fluorocytosine treatment and subsequent regression of the PRObCD+ tumor cells. Immunohistochemical studies of PRObCD+ tumors in animals treated with 5-fluorocytosine found the tumors to be extensively infiltrated with cells that stained for natural killer (NK) antigens. In contrast to the work of other investigators (14-16), few CD8+ cells were seen in the treated lesions, but rather these cells were restricted to the periphery of the tumor. Corticosteroid treatment significantly lowered the number of circulating T cells in the rats but had little effect on the response of the PRObCD+ and wild-type tumors to 5-fluorocytosine, suggesting that the vaccine effect was not directly mediated by CD8+ cells. This contrasts with the work of Consalvo et al. (15), which indicated that monoclonal antibody treatment directed against CD8+ cells reduced the effectiveness of the CD/5-fluorocytosine system. In the present study by Pierrefite-Carle et al. (21), when animals were treated with a monoclonal antibody (anti-asialo GM1) directed against NK cells, the vaccine effect was abrogated, which suggested that NK cells were the primary mediators of the distant bystander effect in this model. Although described previously with the HSV-tk/GCV system, to our knowledge, this report (21) is the first demonstration of a bystander effect at a distance in the CD/5-fluorocytosine enzyme/prodrug system.
The study by Pierrefite-Carle et al. (21) suffers from some limitations. The very small tumors treated in this preclinical model are unlikely to be treated at this stage in the clinic. The 5-fluorocytosine treatments were initiated 24 hours after inoculation, at a time the tumor burden was low. A more realistic approach would have been to see if larger, established tumors, both PRObCD+ and wild-type, could be effectively treated by use of this strategy. The CD/5-fluorocytosine system requires prolonged therapy. In these experiments, continuous 5-fluorocytosine treatment was administered to the animals for 30 days. Evidence supporting an important role for an NK cell antitumor immune responses in suicide gene therapy comes from the work of other investigators (17), who found similar distant bystander effect responses with the HSV-tk/GCV system in strains of mice with severe combined immunodeficiency, which lack T and B cells but which manifest NK cell activity (26). Other workers analyzing the immune response to CD/5-fluorocytosine suicide gene therapy have found the effects to be mediated by CD8+ T lymphocytes and granulocytes (15,27) as well as through the animals' ability to generate a long-lasting immune memory to the tumor, which requires the activity of CD4+ lymphocytes (15). Pierrefiete-Carle et al. found little evidence of a role for the activity of any T cells in their "vaccination" effect. Further work is required to reconcile these different findings.
While the work of Pierrefiete-Carle et al. points out an important direction for systemic gene therapy for cancer, the clinical effectiveness of this strategy is uncertain. The CD/5-fluorocytosine system is already in clinical trials against colorectal (25) and other carcinomas (28). As of yet, there are no results available from these trials. The published results (11,29) of HSV-tk/GCV trials for patients with brain tumors and malignant mesothelioma have been disappointing. Perhaps the level of cellular transduction and/or gene expression in these trials was insufficient to result in adequate cell killing to elicit a vaccine response (30), or perhaps the tumor burdens seen clinically are large enough to render the patient immunologically unresponsive. Melcher et al. (31) have shown that tumor cell killing by suicide genes via apoptotic cell death-independent pathways generates a stronger immune response than cell killing proceeding through apoptotic mechanisms. While tumor vaccination by enzyme/prodrug therapy may hold great promise, for this promise to be realized, this strategy will likely need to be combined with other strategies that increase local tumor killing and antigen exposure. Manipulation of the enzyme/prodrug system, such as by genetic (32) or pharmacologic modifications (33) of gap junctions, by combinations of gene therapy and chemotherapy (34) or radiation therapy (35), or by the use of replication-competent vectors, which would improve tumor transduction through multiple rounds of viral replication and oncolysis (36,37), may help to enhance the potential vaccine effect of this approach.
REFERENCES
1 Morris JC, Touraine R, Wildner O, Blaese RM. Suicide genes: gene therapy applications using enzyme/prodrug strategies. In: Friedman T, editor. The development of human gene therapy. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press; 1999. p. 477-526.
2 Moolten FL. Tumor chemosensitivity conferred by inserted herpes thymidine kinase genes: paradigm for a prospective cancer control strategy. Cancer Res 1986;46:5276-81.[Abstract]
3 Mullen CA, Kilstrup M, Blaese RM. Transfer of the bacterial gene for cytosine deaminase to mammalian cells confers lethal sensitivity to 5-fluorocytosine: a negative selection system. Proc Natl Acad Sci U S A 1992;89:33-7.[Abstract]
4 Freeman SM, Abboud CN, Whartenby KA, Packman CH, Koeplin DS, Moolten FL, et al. The "bystander effect": tumor regression when a fraction of the tumor mass is genetically modified. Cancer Res 1993;53:5274-83.[Abstract]
5 Wildner O, Morris JC, Vahanian NN, Ford H Jr, Ramsey WJ, Blaese RM. Adenoviral vectors capable of replication improve the efficacy of HSVtk/GCV suicide gene therapy of cancer. Gene Ther 1999;6:57-62.[Medline]
6 Bi WL, Parysek LM, Warnick R, Stambrook PJ. In vitro evidence that metabolic cooperation is responsible for the bystander effect observed with HSV tk retroviral gene therapy. Hum Gene Ther 1993;4:725-31.[Medline]
7 Huber BE, Austin EA, Richards CA, Davis ST, Good SS. Metabolism of 5-fluorocytosine to 5-fluorouracil in human colorectal tumor cells transduced with the cytosine deaminase gene: significant antitumor effects when only a small percentage of tumor cells express cytosine deaminase. Proc Natl Acad Sci U S A 1994;91:8302-6.[Abstract]
8 Ishii-Morita H, Agbaria R, Mullen CA, Hirano H, Koeplin DA, Ram Z, et al. Mechanism of `bystander effect' killing in the herpes simplex thymidine kinase gene therapy model of cancer treatment. Gene Ther 1997;4:244-51.[Medline]
9
Mesnil M, Piccoli C, Tiraby G, Willecke K, Yamasaki H.
Bystander killing of cancer cells by herpes simplex virus thymidine kinase gene is mediated by
connexins. Proc Natl Acad Sci U S A 1996;93:1831-5.
10 Dong Y, Wen P, Manome Y, Parr M, Hirshowitz A, Chen L, et al. In vivo replication-deficient adenovirus vector-mediated transduction of the cytosine deaminase gene sensitizes glioma cells to 5-fluorocytosine. Hum Gene Ther 1996;7:713-20.[Medline]
11 Ram Z, Culver KW, Oshiro EM, Viola JJ, DeVroom HL, Otto E, et al. Therapy of malignant brain tumors by intratumoral implantation of retroviral vector-producing cells. Nat Med 1997;3:1354-61.[Medline]
12
Todryk S, Melcher AA, Hardwick N, Lindardakis E, Bateman
A, Colombo MP, et al. Heat shock protein 70 induced during tumor cell killing induces Th1
cytokines and targets immature dendritic cell precursors to enhance antigen uptake. J
Immunol 1999;163:1398-408.
13 Caruso M, Panis Y, Gagandeep S, Houssin D, Saltzmann JL, Klatzmann D. Regression of established macroscopic liver metastases after in situ transduction with a suicide gene. Proc Natl Acad Sci U S A 1993;90:7024-8.[Abstract]
14 Barba D, Hardin J, Sadelain M, Gage FH. Development of anti-tumor immunity following thymidine kinase-mediated killing of experimental brain tumors. Proc Natl Acad Sci U S A 1994;91:4348-52.[Abstract]
15
Consalvo M, Mullen CA, Modesti A, Musiani P, Allione A,
Cavallo F, et al. 5-Fluorocytosine-induced eradication of murine adenocarcinomas engineered to
express the cytosine deaminase suicide gene requires host immune competence and leaves an
efficient memory. J Immunol 1995;154:5302-12.
16 Yamamoto S, Suzuki S, Hoshino A, Akimoto M, Shimada T. Herpes simplex virus thymidine kinase/ganciclovir-mediated killing of tumor cell induces tumor-specific cytotoxic T cells in mice. Cancer Gene Ther 1997;4:91-6.[Medline]
17
Dilber MS, Abedi MR, Bjorkstrand B, Christensson B, Gahrton
G, Xanthopoulos KG, et al. Suicide gene therapy for plasma cell tumors. Blood 1996;88:2192-200.
18 Wei MX, Bougnoux P, Sacre-Salem B, Peyrat MB, Lhuillery C, Salzmann JL, et al. Suicide gene therapy of chemically induced tumor in rat: efficacy and distant bystander effect. Cancer Res 1998:58:3529-32.[Abstract]
19 Gagandeep S, Brew R, Green B, Christmas SE, Klatzmann D, Poston GJ, et al. Prodrug-activated gene therapy: involvement of an immunological component in the "bystander effect." Cancer Gene Ther 1996;3: 83-8.[Medline]
20 Ramesh R, Munshi A, Abboud CN, Marrogi AJ, Freeman SM. Expression of costimulatory molecules: B7 and ICAM up-regulation after treatment with a suicide gene. Cancer Gene Ther 1996;3:373-84.[Medline]
21
Pierrefite-Carle V, Baque P, Gavelli A, Mala M, Chazal M,
Gugenheim J, et al. Cytosine deaminase/5fluorocytosine-based vaccination against liver tumors:
evidence of a distant bystander effect. J Natl Cancer Inst 1999;91:2014-9.
22 Polak A, Eschenhof E, Fernex M, Scholer HJ. Metabolic studies with 5-fluorocytosine-14C in mouse, rat, rabbit, dog and man. Chemotherapy 1976;22:137-53.[Medline]
23 Austin EA, Huber BE. A first step in the development of gene therapy for colorectal carcinoma: cloning, sequencing, and expression of Escherichia coli cytosine deaminase. Mol Pharmacol 1993;43:380-7.[Abstract]
24 Ge K, Xu L, Zheng Z, Xu D, Sun L, Liu X. Transduction of cytosine deaminase gene makes rat glioma cells highly sensitive to 5-fluorocytosine. Int J Cancer 1997;71:675-9.[Medline]
25 Crystal RG, Hirschowitz E, Lieberman M, Daly J, Kazam E, Henschke C, et al. Phase I study of direct administration of a replication deficient adenovirus vector containing the E. coli cytosine deaminase gene to metastatic colon carcinoma of the liver in association with the oral administration of the pro-drug 5-fluorocytosine. Hum Gene Ther 1997;8:985-1001.[Medline]
26 Budzynski W, Radzikowski C. Cytotoxic cells in immunodefficient athymic mice. Immunopharmacol Immunotoxicol 1994;16:319-46.[Medline]
27 Kuriyama S, Kikukawa M, Masui K, Okuda H, Nakatani T, Sakamoto T, et al. Cytosine deaminase/5-fluorocytosine gene therapy can induce efficient anti-tumor effects and protective immunity in immunocompetent mice but not in athymic nude mice. Int J Cancer 1999;81:592-7.[Medline]
28
Roth JA, Cristiano RJ. Gene therapy for cancer: what have we
done and where are we going? J Natl Cancer Inst 1997;89:21-39.
29 Sterman DH, Treat J, Litzky LA, Amin KM, Coonrod L, Molnar-Kimber K, et al. Adenovirus-mediated herpes simplex virus thymidine kinase/ganciclovir gene therapy in patients with localized malignancy: results of a phase I clinical trial in malignant mesothelioma. Hum Gene Ther 1998;9:1083-92[Medline]
30 Vile RG, Castleden S, Marshall J, Camplejohn R, Upton C, Chong H. Generation of an anti-tumour immune response in a non-immunogenic tumour: HSVtk killing in vivo stimulates a mononuclear cell infiltrate and a Th1-like profile of intratumoural cytokine expression. Int J Cancer 1997;71:267-74.[Medline]
31 Melcher A, Todryk S, Hardwick N, Ford M, Jacobson M, Vile RG. Tumor immunogenicity is determined by the mechanism of cell death via induction of heat shock protein expression. Nat Med 1998;4:581-7.[Medline]
32 Touraine RL, Ishii-Morita H, Ramsey WJ, Blaese RM. The bystander effect in the HSVtk/ganciclovir system and its relationship to gap junctional communication. Gene Ther 1998;12:1705-11.
33 Touraine RL, Vahanian N, Ramsey WJ, Blaese RM. Enhancement of the herpes simplex virus thymidine kinase/ganciclovir bystander effect and its antitumor efficacy in vivo by pharmacologic manipulation of gap junctions. Hum Gene Ther 1998;9:2385-91.[Medline]
34 Wildner O, Blaese RM, Morris JC. Synergy between the herpes simplex virus-tk/ganciclovir prodrug suicide system and the topoisomerase I inhibitors topotecan. Hum Gene Ther. In press 1999.
35 Khil MS, Kim JH, Mullen CA, Kim SH, Freytag SO. Radiosenstization by 5-fluorocytosine of human colorectal carcinoma cells transduced with cytosine deaminase gene. Clin Cancer Res 1996;2:53-7.[Abstract]
36
Heise CC, Williams AM, Xue S, Propst M, Kirn DH.
Intravenous administration of ONYX-015, a selectively replicating adenovirus, induces
antitumoral efficacy. Cancer Res 1999;59:2623-8.
37
Wildner O, Blaese RM, Morris JC. Therapy of colon cancer with
oncolytic adenovirus is enhanced by the addition of herpes simplex virus-thymidine kinase. Cancer Res 1999;59:410-3.
This article has been cited by other articles in HighWire Press-hosted journals:
![]() |
||||
|
Oxford University Press Privacy Policy and Legal Statement |