The role of cellular- and prodrug-associated factors in the bystander effect induced by the Varicella zoster and Herpes simplex viral thymidine kinases in suicide gene therapy

Abstract

To investigate the factors influencing the bystander effect Ð a key element in the efficacy of suicide gene therapy against cancer Ð we compared the effect triggered by four extremely efficient gene / prodrug combinations, i.e., VZVtk / BVDU, the thymidine kinase of Varicella zoster virus associated with ( E ) -5 -( 2 -bromovinyl ) -2 H -deoxyuridine; VZVtk / BVaraU, the same enzyme associated with ( E ) -5 -( 2 -bromovinyl ) -1 --D -arabinofuranosyluracil; HSVtk / BVDU, the association of the Herpes simplex virus thymidine kinase with BVDU; and the classical HSVtk / GCV ( ganciclovir ) paradigm. The cells used, the human MDA -MB -435 breast cancer, and the rat 9L glioblastoma lines were equally sensitive in vitro to these four associations. In both cell types, the combinations involving pyrimidine analogues ( BVDU, BVaraU ) displayed a smaller bystander killing than the combination involving the purine analogue ( GCV ) . In addition, the bystander effect induced by all the tk / prodrug systems was reduced in MDA -MB -435 cells in comparison to 9L cells; albeit, the viral kinases were produced at a higher level in the breast cancer cells. All systems induced apoptotic death in the two cell types, but the MDA -MB -435 cells, deprived of connexin 43, were noncommunicating in striking contrast with the 9L cells. That functional gap junctions have to be increased in order to improve the breast cancer cell response to suicide gene therapy was demonstrated by transducing the Cx43 gene: this modification enhanced the bystander effect associated in vitro with GCV treatment and, by itself, decreased the tumorigenicity of the untreated cells. However, the noncommunicating MDA -MB -435 cells triggered a significant bystander effect both in vitro and in vivo with the HSVtk / GCV system, showing that communication through gap junctions is not the only mechanism involved. Cancer Gene Therapy ( 2000 ) 7, 1456 ± 1468Key words: Suicide gene therapy; viral thymidine kinases; bystander effect; connexin 43.G ene therapy is a promising approach for the treatment of human cancers. The introduction into tumoral cells of à`s uicide'' gene, whose product is able to activate a nontoxic prodrug into a toxic compound, is a powerful method to selectively kill the modified cells. The most frequently used system consists of transferring the Herpes simplex thymidine kinase gene ( HSVtk ) into tumor cells and to treat them with ganciclovir (GCV ) . This guanosine analogue is specifically monophosphorylated by the viral kinase and then converted by cellular enzymes into the triphosphate derivative, which, upon incorporation into elongating DNA, induces cell death by premature chain termination. The efficacy of the HSVtk / GCV system first described by Moolten 1 in 1986 has been demonstrated in many different types of tumoral cells in vitro and in vivo, and it is currently under clinical investigation as treatment for a wide variety of cancers.A major obstacle to successful cancer gene therapy is the poor efficiency of the gene transfer process r...

Functional expression of thymidine kinase in human leukaemic and colorectal cells, delivered as EGFP fusion protein by herpesvirus saimiri-based vector

“…This is supported by our observation that GCV has a greater effect than BVDU, which correlates with GCV being known to have a greater bystander effect than BVDU. 47,9 It has also been proposed that the host immune system may play a major role in in vivo bystander killing.…”

“…1 In in vitro settings BVDU is a more efficient and less toxic prodrug than GCV, 7,8 but has a much less potent bystander effect than GCV in HSV-TK-transduced human glioma cells. 9,10 Gene delivery systems that are currently being evaluated by clinical trials include retroviruses, lentiviruses, foamiviruses, adeno and adenoassociated viruses, herpesviruses and cationic liposomes/DNA-protein complexes. So far, TK gene has been delivered by retroviruses and adenoviruses in in vitro [11][12][13][14] and in vivo [15][16][17] applications.…”

Sufasalazine unveils a contact-independent HSV-TK/ganciclovir gene therapy bystander effect in malignant gliomas

“…Clones LN18-TK, C6-TK and U87-TK were obtained by transfection with a defective murine retrovirus carrying the HSV-tk and the geneticin resistance genes (gift of Dr C. Grignet), selection in G418, and clonal expansion and selected on the basis of their sensitivity to ganciclovir and of their growth characteristics similar to those of parental LN18, C6 and U87 cells, respectively (20,21). Clone 9L-TK was a gift from Dr C. Grignet (22). All clones were kept in RPMI-C or DEM-C supplemented with G418.…”

Gene therapy for carcinoma of the breast

“…The enzymes proposed for GDEPT for breast cancer can be divided into two categories. The first category consists of 'foreign' enzymes of nonmammalian origin, such as viral TK, 80,81 bacterial cytosine deaminase (CD), and carboxypeptidase G2 (CPG2). 79,82,83 The second category comprises enzymes of human origin, such as cytochrome P450 isophorms.…”