Double Suicide Gene (Cytosine Deaminase and Herpes Simplex Virus Thymidine Kinase) but Not Single Gene Transfer Allows Reliable Elimination of Tumor CellsIn Vivo

Abstract

Suicide genes such as cytosine deaminase (CD) and herpes simplex virus thymidine kinase (TK) encode products that convert nontoxic substances (prodrugs) into toxic metabolites. Suicide gene transfer is currently being used in cancer therapy or can be used as a safety modality. To analyze the reliability of suicide genes as a safety modality for a vaccination study with viable cytokine/B7 gene-modified tumor cells, the individual and combined efficacy of the two suicide genes was compared for in vitro and in vivo cell killing of a murine mammary adenocarcinoma cell line (TS/A). To adapt the system to an in vivo gene delivery situation, bulk cultures cotransfected with the CD and TK gene were used instead of selected clones. In vitro, both CD and TK conferred sensitivity to the respective prodrug but the combined cytotoxic effects of both gene products were always superior. For in vivo analysis BALB/c mice were injected subcutaneously with CD- and TK-modified TS/A cells, treated with prodrugs, and tumor size was evaluated for a period of 100 days. In the in vivo situation the combination of both enzyme/prodrug systems was again most effective. The highest single concentration of 5-FC (500 mg/kg) or GCV (100 mg/kg) was not able to fully protect the animals from developing tumors, whereas a combination of 5-FC (250 mg/kg) and GCV (50 mg/kg) resulted in complete tumor eradication. In nude mice treated in the same way, most CD/TK tumors could not be eliminated. Furthermore, BALB/c mice cured of TS/A-CD/TK tumors developed a systemic tumor immunity against challenge with parental TS/A cells. These findings indicate that reliable tumor elimination by the suicide genes depends on T cells. The cooperative effect of both suicide genes was confirmed in vitro with the human renal cell carcinoma line RCC26. We conclude that TK and CD together, but neither gene alone, act as a safety mechanism for the elimination of tumor cells in a reliable fashion and suggest that a rapid and quantitative antigen release by effective TK- and CD-mediated tumor destruction is necessary for T cell immunity to develop.

TRAIL enhances thymidine kinase/ganciclovir gene therapy of neuroblastoma cells

“…Cells were cultured in RPMI 1640 medium ( Life Technologies, Eggenstein, Germany ) supplemented with 10% heat -inactivated FCS (Conco, Wiesbaden, Germany ), 10 mM HEPES, pH 7.3 (Biochrom, Berlin, Germany ), 100 U /mL penicillin (Life Technologies ), 100 g /mL streptomycin ( Life Technologies ), 2 mM L -glutamine (Biochrom ), 0.5 mg /mL G418 (Life Technologies), or 0.5 mg /mL hygromycin ( Life Technologies ), as previously described. 2,14,44 Clonogenicity assay 2Â10 4 TK + or TK À cells per well were plated into 12-well plates, allowed to attach overnight, and then treated with GCV in indicated concentrations for 4 days. Cells still adherent were trypsinized and pooled with the cells already detached.…”

“…34 Apoptosis can start from two different pathways -the death receptor pathway and the mitochondrial pathwaywith both pathways eventually converging to activate effector caspases. 1,37,44,49 TK / GCV -induced apoptosis in SH -EP neuroblastoma cells is triggered by ligand -independent aggregation of CD95, which recruits the adaptor molecule FADD and caspase -8, forming a death -inducing signaling complex ( DISC ). 2 Caspase-8 thus becomes activated and in turn activates downstream effector caspases by proteolytic cleavage, leading to apoptosis.…”

Enhanced suicide gene effect by adenoviral transduction of a VP22-cytosine deaminase (CD) fusion gene

“…[14][15][16][17] A comparison of the TK/GCV with the CD/5-FC approach in cell culture 18 and implanted tumours 19 suggested that the cytosine deaminase system might be superior, since its bystander effect does not depend on gap junctional intercellular communication channels.…”

Enhanced growth suppression in esophageal carcinoma cells using adenovirus-mediated fusion gene transfer (uracil phosphoribosyl transferase and herpes simplex virus thymidine kinase)

“…A bystander effect of AxCA.UT gene therapy may be useful for such solid tumor treatment. Important advantages of combination gene therapy were reported by Rogulski et al 12 and Uckert et al 13 as a synergistic effect in cytosine deaminase plus TK therapy. However, in AxCA.UT infection with 5-FU /GCV treatment, an additive, rather than a synergistic effect, was observed in human esophageal cancer cells compared to AxCA.UP infection with 5 -FU treatment.…”

“…Furthermore, it has been reported that the use of cytokine genes or prodrug gene, IL -12, 30 GM -CSF, 31 or cytosine deaminase, 12,13,32 combined with the TK /GCV system, can reinforce the effect of the TK /GCV system. Similarly, we might expect the same reinforcement of the effect of UT with the 5-FU /GCV system when adding the cytosine deaminase gene into the construct.…”