The equine herpes virus 4 thymidine kinase is a better suicide gene than the human herpes virus 1 thymidine kinase

Loubière, Laurence; Tiraby, Michele; Cazaux, Christophe; Brisson, E.; Grisoni, Michel; Zhao‐Emonet, Jing Chao; Tiraby, Gérard; Klatzmann, David

doi:10.1038/sj.gt.3300993

Cited by 21 publications

(18 citation statements)

References 20 publications

(17 reference statements)

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“…11 There have, therefore, been many attempts to improve the efficacy of tk /GCV. These have included the use of the tk gene from other herpes viruses, 12,13 isolation of HSV1 tk mutants better able to activate GCV, 14 the use of alternative prodrugs, 15 combining tk /GCV with other GPAT strategies, such as cytosine deaminase / 5-fluorocytosine, 16 and combined GPAT-immunotherapy strategies, via delivery of the genes for both tk and cytokines such as interleukins - 2 17 and -4. 18 Recently, there has been some elucidation of the pathways by which activated GCV induces apoptosis in transfected cells.…”

mentioning

confidence: 99%

Herpes simplex virus thymidine kinase/ganciclovir–induced cell death is enhanced by co-expression of caspase-3 in ovarian carcinoma cells

et al. 2001

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There is a need to enhance the efficacy of genetic prodrug activation therapy using herpes simplex virus thymidine kinase ( tk ) and ganciclovir ( GCV ) following disappointing results in early clinical trials. tk / GCV has been shown to lead to the activation of caspase -3, a potent executor of apoptosis. We demonstrate that co -expression of pro -caspase -3 with tk / GCV leads to enhanced cell death in ovarian carcinoma cells in vitro. Following transfection with recombinant adenoviral vectors encoding tk, GCV treatment leads to greater cell death in pro -caspase -3 ± expressing clones of SKOV3 and IGROV1 than control cells, as well as more rapid activation of caspase -3 and more rapid cleavage of PARP. Flow cytometry suggests that there is a greater degree of S -phase block in the pro -caspase -3 ± expressing clones than in control cells following treatment with tk / GCV. None of these effects is seen following transfection with a control adenovirus that does not encode tk. The increased cell death, early caspase -3 activation and PARP cleavage, and flow cytometric changes seen in pro -caspase -3 ± expressing cells can be partially inhibited by treatment with benzyloxycarbonyl ± val ± ala ± asp fluoromethylketone, a synthetic caspase inhibitor. Our data suggest that co -expression of procaspase -3 may lead to a significant enhancement of the efficacy of tk / GCV therapy. Cancer Gene Therapy ( 2001 ) 8, 308 ± 319

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confidence: 99%

Herpes simplex virus thymidine kinase/ganciclovir–induced cell death is enhanced by co-expression of caspase-3 in ovarian carcinoma cells

et al. 2001

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“…The viral DNA polymerase incorporates the metabolites preferentially into the elongating viral DNA, resulting in DNA-chain termination [4,5]. Recently, the herpesviral TK, such as those of HSV (herpes simplex virus), VZV (Varicella Zoster virus), equine herpes virus 4 and EBV (Epstein-Barr virus) [6][7][8][9] also have been found to be useful suicide genes in gene therapy. The genes are introduced into tumour cells, which, subsequently, can be killed selectively by the appropriate nucleoside analogues.…”

Section: Introductionmentioning

confidence: 99%

Identification and characterization of the conserved nucleoside-binding sites in the Epstein-Barr virus thymidine kinase

Chen

Chang

et al. 2004

Biochemical Journal

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Thymidine kinase (TK), encoded by EBV (Epstein-Barr virus), is an attractive target for antiviral therapy and provides a novel approach to the treatment of EBV-associated malignancies. Despite the extensive use of nucleoside analogues for the treatment of viral infections and cancer, the structure-function relationship of EBV TK has been addressed rarely. In the absence of any structural information, we sought to identify and elucidate the functional roles of amino acids in the nucleoside-binding site using site-directed mutagenesis. Through alignment with other human herpesviral TK protein sequences, we predicted that certain conserved regions comprise the nucleoside-binding site of EBV TK and, through site-directed mutagenesis, showed significant changes in activity and binding affinity for thymidine of site 3 (-DRH-) and 4 (-VFP-) mutants. For site 3, only mutants D392E (Asp 392 → Glu) and R393H retain activity, indicating that a negative charge is important for Asp 392 and a positive charge is required for Arg 393 . The increased binding affinities of these two mutants for 3 -deoxy-2 ,3 -didehydrothymidine suggest that the two residues are also important for substrate selection. Interestingly, the changed metal-ion usage pattern of D392E reveals that Asp 392 plays multiple roles in this region. His 394 cannot be compensated by other amino acids, also indicating a crucial role. In site 4, the F402Y mutant retains full activity; however, F402S retains only 60 % relative activity. Strikingly, when Phe 402 is substituted with serine residue, the original preferred pyrimidine substrates, such as 3 -azido-3 -deoxythymidine, iododeoxyuridine and β-L-5-iododioxolane uracil (L-form substrate), have decreased competitiveness with thymidine, suggesting that Phe 402 plays a crucial role in substrate specificity and that the aromatic ring is important for function.

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“…Our study aimed to modify the catalytic activity of another member or the herpes thymidine kinase family, the equine herpes virus-4 thymidine kinase (EHV4 TK). Previous research found that a human osteosarcoma cell-line expressing EHV4 TK was rendered 12-fold more sensitive to GCV compared to the same cell-line expressing HSV1 TK [23]. Therefore, in line with the studies focusing on HSV1 TK protein engineering, we investigated if it is possible to further improve EHV4 TK/GCV cytotoxicity by designed enzyme variants.…”

Section: Discussionmentioning

confidence: 99%

“…The plasmids pUT599[HSV1 TK] and pUT699[EHV4 TK] have been described previously [23]. A maltose-binding protein (MBP)-polyhistidine double affinity fusion of EHV4 TK was obtained by cloning into a modified pMAL c2 vector (New England Biolabs, Ipswich, MA) a fragment of two annealed oligos (5 0 -cgc acc atc acc atc acc acc agg tcg gtg gtg gcg gtg gct tgg ttc cgc gtg gct ctc ata tgg aat tcg-3 0 , and 5 0 -gat ccg aat tcc ata tga gag cca cgc gga acc aag cca ccg cca cca ccg acc tgg tgg tga tgg tga tgg tgc gag ct-3 0 ) to generate a linker coding for the amino acid sequence HHHHHHQVGGGGGLVPRGS with an N-terminal SacI site and C-terminal NdeI, EcoRI and BamHI sites.…”

Section: Molecular Biologymentioning

confidence: 99%

“…Previously, Loubiè re et al compared thymidine kinases expressed by other members of the herpes virus family and found that both murine and human cell-lines transfected with EHV4 TK, which is expressed by the equine herpes virus type-4, resulted in 3-12 times more GCV-induced cytotoxicity compared to HSV1 TKtransfected cells [23]. Based on the EHV4 TK crystal structure and sequence homologies to HSV1 TK, we have generated EHV4 TK mutants also with the aim to either ablate or reduce the affinity of dT binding and ultimately increase the GCV-induced cell killing sensitivity [24].…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

A designed equine herpes thymidine kinase (EHV4 TK) variant improves ganciclovir-induced cell-killing

McSorley

Ort

Monnerjahn

et al. 2014

Biochemical Pharmacology

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The equine herpes virus 4 thymidine kinase is a better suicide gene than the human herpes virus 1 thymidine kinase

Cited by 21 publications

References 20 publications

Herpes simplex virus thymidine kinase/ganciclovir–induced cell death is enhanced by co-expression of caspase-3 in ovarian carcinoma cells

Herpes simplex virus thymidine kinase/ganciclovir–induced cell death is enhanced by co-expression of caspase-3 in ovarian carcinoma cells

Identification and characterization of the conserved nucleoside-binding sites in the Epstein-Barr virus thymidine kinase

A designed equine herpes thymidine kinase (EHV4 TK) variant improves ganciclovir-induced cell-killing

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