Tat8–TK/GCV Suicide Gene Therapy Induces Pancreatic Tumor Regression In Vivo

Abstract

Suicide gene therapy using the herpes simplex virus thymidine kinase (TK) gene in combination with ganciclovir (GCV) has been shown to produce therapeutic, but limited, efficacy because of poor gene transfer efficiency and reduced bystander effect. Here we report that fusion of TK to an eight-amino acid peptide from the basic domain of the human immunodeficiency virus (HIV) Tat protein significantly increases the cytotoxic efficacy of the TK/GCV system in pancreatic cancer cells. We demonstrate that Tat8-TK protein is released from the intracellular compartment of Tat8-TK-expressing cells to the extracellular medium after GCV treatment. Interestingly, we show that this conditioned medium is then able to mediate cytotoxicity of wildtype cultures, suggesting the internalization of the Tat8-TK protein. Moreover, a strong antitumoral effect of Tat8-TK/GCV treatment could be achieved by two different in vivo approaches. Tumors injected with NIH 3T3/Tat8-TK cells attached to microcarriers (MC+Tat8-TK) and treated with GCV led to a 35.6% reduction in the initial tumor volume and to 50% tumor eradication. Furthermore, electrogene transfer of TK or Tat8-TK followed by administration of high doses of GCV led to an overall statistically significant reduction in tumor growth. However, the reduction in initial tumor volume was statistically significant only for the Tat8-TK group (59.5% reduction). Moreover, in this group 50% complete tumor eradication was achieved. When moderate doses of GCV were administered, the overall reduction in tumor growth was statistically significant only in the Tat8-TK group. Therefore, our results suggest that fusion of TK to the Tat8 peptide enhances TK/GCV suicide gene therapy.

E-cadherin contributes to the bystander effect of TK/GCV suicide therapy and enhances its antitumoral activity in pancreatic cancer models

“…To such end, we generated a recombinant adenovirus, AdTat8TKIE, that co-expresses both, a modified TK, previously shown to have enhanced cytotoxicity, 16 and human E-cadherin, and a control virus, AdTat8TK, which only expresses the modified TK gene (Figure 5a). To confirm the functionality of the viruses, we transduced NP-18, BxPC-3, PANC-1, MIA PaCa-2 and NP-9 pancreatic ductal adenocarcinoma cell lines with different doses of AdTat8TKIE or AdTat8TK, and analyzed E-cadherin expression after 48 h. Immunoblot detection revealed E-cadherin overexpression in all the cell lines transduced with the AdTat8TKIE virus ( Figure 5b).…”

“…The AdTat8TK recombinant adenovirus was generated by inserting the 1.2 kb sequence of the Tat8TK gene, a modified TK with enhanced cytotoxicity, 16,34 into the BglII/XhoI sites of the pShuttleCMV plasmid (Addgene, Cambridge, MA, USA). The AdTat8TKIE adenovirus was generated by introducing an IRES element at the XhoI site of the pshuttleCMVTat8TK and the E-cadherin gene at the EcoRV site.…”

“…The oligonucleotides were annealed and cloned into SalI/XbaI-digested pU6 vector, as previously described. 38 The expression cassette for the fusion protein Tat8TK 16 was cloned in a pShuttle-CMV plasmid (Addgene), between the Bgl II and Sal I restriction sites. The U6 promoter and the region encoding the shRNA against Bcl-2 or an shRNA against a nontarget sequence (shCt) 39 were cloned downstream of the Tat8TK expression cassette, in an inverse orientation, at the NotI HindIII sites.…”

Characterization of HIV-1 TAT peptide as an enhancer of HSV-TK/GCV cancer gene therapy

“…On the other hand, TAT-mediated enhancement of cell killing in HSV-TK/GCV therapy has been shown in vitro and in vivo in other studies. [32][33][34] Furthermore, in addition to HSV-TK/GCV therapy, the enhancement has been demonstrated using CYP2B1 suicide gene combined to cyclophosamide, which also induces cell death via apoptosis. 34 One feature of the full-length TAT protein is the ability to suppress host cell's immune response to interfere with the antiviral actions of interferons (IFNs).…”

“…[32][33][34] Furthermore, in addition to HSV-TK/GCV therapy, the enhancement has been demonstrated using CYP2B1 suicide gene combined to cyclophosamide, which also induces cell death via apoptosis. 34 One feature of the full-length TAT protein is the ability to suppress host cell's immune response to interfere with the antiviral actions of interferons (IFNs). 35,36 To rule out that the enhancement of the cell killing in certain cell lines was a consequence of capability of TAT peptide to suppress innate immune response as well, we studied the contribution of the type I IFN response to transduction with TAT-TK-GFP and TK-GFP vectors.…”