Purified herpes simplex virus thymidine kinase retroviral particles:

Abstract

An important consequence of the suicide gene therapeutic paradigm is the phenomenon of bystander cell killing, the death of adjacent tumor cells not transduced with the thymidine kinase ( TK ) gene from herpes simplex virus ( HSV ) after treatment with the antiviral drug, ganciclovir ( GCV ). Evidence from quantitative in vitro assays of glioma cell lines suggest that both murine and human gliomas are similar in expressing high sensitivity to the bystander effect. In five of six glial tumors examined, the presence of only 5% of HSV -TK -expressing transduced cells in the culture resulted in > 90% tumor cell death / stasis after addition of GCV. Several lines of evidence support gap junction intercellular communication ( GJIC ) as important in the bystander effect. In vitro metabolic assays, performed with GCV in the medium, indicated that more tumor burden was reduced when culture conditions supported cell -cell contact of parental and HSV -TK -transduced cells. Additionally, a double dye transfer assay showed that cell communication through the gap junction is greatest for glioma, less for melanoma, and much less for colorectal carcinoma cell lines. In vitro metabolic assays with mixtures of TK + / TK À homologous tumor cells confirmed that glioma cell lines were more susceptible to bystander killing than melanomas. Assays with chimeric tumor mixtures of TK + / TK À cells showed that the level of the bystander killing obtained was characteristic of the TK À bystander cells. The in vitro findings were confirmed in vivo with GCV -treated homologous and chimeric tumors composed of TK + / TK À cells. Day 21 mean tumor volumes ( MTVs ) indicated the growths obtained were characteristic of the bystander activity reflective of the nontransduced cell population. Furthermore, nontransduced, high -GJIC cells in a chimeric tumor mass appeared to effectively bridge between transduced tumor cells and poorly communicating nontransduced cells. Finally, the importance of a gap junction protein, such as connexin -43, in facilitating the bystander effect was demonstrated with the HT29 low -GJIC cell line. When the TK -nontransduced cell population expressed connexin -43, a better bystander kill was achieved compared to the parental counterpart.

Acyclovir Is Activated into a HIV-1 Reverse Transcriptase Inhibitor in Herpesvirus-Infected Human Tissues

“…Apparently, in reconstituted MT4 cultures ACV that is phosphorylated in HHV-6-infected cells is transferred to HIV-1-infected cells, since the majority of these cells were not coinfected with HHV-6. These results are in full agreement with the published data on the transfer of phosphorylated ACV between cells (Burrows et al, 2002; Degreve et al, 1999). In tissues, transfer of phosphorylated ACV between cells is facilitated by specialized contacts (Nicholas et al, 2003).…”

A heterotypic bystander effect for tumor cell killing after adeno-associated virus/phage–mediated, vascular-targeted suicide gene transfer

“…The intercellular junctions formed by both endothelial cells and tumor parenchymal cells contain Cx43 and Cx26; overexpression of which in gap junctions have been shown to potentiate the bystander effect (33, 34, 40). Consequently, gene transfer-mediated forced expression of these connexins in cells with low levels of gap junctions can result in potent induction of a bystander effect in cells lacking expression of the suicide gene (41). Therefore, the expression of these connexins selectively in the cell types displaying bystander effects in this study suggests that these proteins are involved in the heterotypic bystander killing described here.…”

“…Therefore, the expression of these connexins selectively in the cell types displaying bystander effects in this study suggests that these proteins are involved in the heterotypic bystander killing described here. It is generally assumed that the level of the bystander effect is determined by the characteristics of the non- HSVtk -transduced cell population (40, 41). Consistent with this assumption, we did not observe bystander killing in co-cultures of connexin-expressing HSVtk -transduced endothelial cells and non-connexin-expressing, non-transduced MDA-MB435 cells.…”

Brainstem Tumors: Where Are We Today?

“…Uncontrolled cellular proliferation, lack of apoptosis, invasion, and angiogenesis are among the biological processes that make these tumors both aggressive and difficult to treat, and hence genetic therapy directed at such process may prove efficacious. Prodrug activation systems using the herpes simplex virus thymidine kinase (HSV-TK) gene and treatment with ganciclovir (GCV) is the most widely studied cancer gene therapy [64, 65]. HSV-TK converts the prodrug GCV into a toxic nucleotide analogue, whose incorporation into cellular DNA blocks cell proliferation.…”