A Truncated Herpes Simplex Virus Thymidine Kinase Phosphorylates Thymidine and Nucleoside Analogs and Does Not Cause Sterility in Transgenic Mice

Abstract

Dividing eukaryotic cells expressing the herpes simplex virus type 1 thymidine kinase (TK) gene are sensitive to the cytotoxic effect of nucleoside analogs such as acyclovir or ganciclovir (GCV). Transgenic mice with cell-targeted expression of this conditional toxin have been used to create animals with temporally controlled cell-specific ablation. In these animal models, which allow the study of the physiological importance of a cell type, males are sterile. In this study, we showed that this phenomenon is due to testis-specific high-level expression of short TK transcripts initiated mainly upstream of the second internal ATG of the TK gene. This expression is DNA methylation independent. To obtain a suicide gene that does not cause male infertility, we generated and analyzed the properties of a truncated TK (⌬TK) lacking the sequences upstream of the second ATG. We showed that when expressed at sufficient levels, the functional properties of ⌬TK are similar to those of TK in terms of thymidine or GCV phosphorylation. This translated into a similar GCV-dependent toxicity for ⌬TK-or TK-expressing cells, both in vitro and in transgenic mice. However, ⌬TK behaved differently from TK in two ways. First, it did not cause sterility in ⌬TK transgenic males. Second, low-level ⌬TK RNA expression did not confer sensitivity to GCV. The uses of ⌬TK in cell-specific ablation in transgenic mice and in gene therapy are discussed.The herpes simplex virus type 1 thymidine kinase (TK) gene has gained considerable importance in clinical medicine and as a research tool. TK can substitute for cellular thymidine kinase in the metabolic pathway of thymidine, and eukaryotic cells lacking functional TK can be rescued from culture in hypoxanthine-aminopterin-thymidine (HAT) selection medium by TK expression (44). Furthermore, TK can phosphorylate certain nucleoside analogs that are not metabolized by cellular enzymes (21). This property has allowed the discovery of nucleoside analogs such as acyclovir and ganciclovir (GCV) that possess strong activity against herpesvirus infections (19,21). This same property turned out to be useful for negative selection of TK-expressing cells. Phosphorylated-nucleoside analogs such as acyclovir triphosphate or GCV triphosphate are potent toxic metabolites for dividing cells. They are incorporated into elongating DNA by cellular DNA polymerase and induce chain termination and eventually cell death (20,36,42).This conditional toxicity of TK can be utilized in vivo. TKmediated destruction of undesirable cells is being developed for gene therapy of cancer or human immunodeficiency virus (HIV) infection (10,11,15,36). Effectiveness has been demonstrated in animal models, and clinical trials for cancer gene therapy have already been initiated (14). In addition, Heyman et al. developed the concept of TK obliteration, the ablation of a specific cell type in transgenic mice (26). This conditional cell knockout is obtained by expressing TK with a cell-specific promoter. This system offers the valuable advantag...

Atrophy of brown adipocytes in the adult mouse causes transformation into white adipocyte-like cells

“…Among three UCP-TK founder mice, two female mice exhibited germ-line transmission of the transgene whereas a male founder was sterile, consistent with previous reports that transgenic mice harboring the HSV-TK gene produce abnormal sperms due to a testis specific extra transcription initiation site within the HSV-TK gene (Robert et al, 1990;Salomon et al, 1995; see also Figure 1B). The copy number of the transgene was estimated to be approximately 5 by genomic Southern blotting using UCP1 and HSV-TK gene probes and genomic DNA prepared from various tissues from transgenic mice (data not shown).…”

FSP1+ Fibroblasts Promote Skin Carcinogenesis by Maintaining MCP-1-Mediated Macrophage Infiltration and Chronic Inflammation

“…18 -20 With activation of the "suicide" gene TK, the phosphorylated products of ganciclovir (GCV) 21 are able to deplete the proliferating FSP1 ϩ cells in vivo selectively. In the current study, we showed for the first time that depletion of proliferating fibroblasts during the tumor promotion stage decreased the incidence and number of papillomas as well as their malignant conversion rate.…”

Adenovirus-mediated expression of HSV1-TK or Fas ligand induces cell death in primary human glioma-derived cell cultures that are resistant to the chemotherapeutic agent CCNU

“…Toxicity due to HSV1 -TK expression has also been seen in the pituitary tumor cell line GH3 35 and in transgenic male mice expressing HSV1 -TK. 42 The second gene therapy approach tested was the RAd delivery of mFasL. Four of the five cell cultures tested were killed by this treatment method.…”