TK Expression Research Articles

Provocative gene therapy strategy for the treatment of hepatocellular carcinoma.

An approach involving retroviral–mediated gene therapy for the treatment of neoplastic disease is described. This therapeutic approach is called “virus–directed enzyme/prodrug therapy” (VDEPT). The VDEPT approach exploits the transcriptional differences between normal and neoplastic cells to achieve selective killing of neoplastic cells. We now describe development of the VDEPT approach for the treatment of hepatocellular carcinoma. Replication–defective, amphotrophic retroviruses were constructed containing a chimeric varicella–zoster virus thymidine kinase (VZV TK) gene that is transcriptionally regulated by either the hepatoma–associated α–fetoprotein or liver–associated albumin transcriptional regulatory sequences. Subsequent to retroviral infection, expression of VZV TK was limited to either α–fetoprotein or albumin–positive cells, respectively. VZV TK metabolically activated the nontoxic prodrug 6–methoxypurine arabinonucleoside (araM), ultimately leading to the formation of the cytotoxic anabolite adenine arabinonucleoside triphosphate (araATP). Cells that selectively expressed VZV TK became selectively sensitive to araM due to the VZV TK–dependent anabolism of araM to araATP. Hence, these retroviral–delivered chimeric genes generated tissue–specific expression of VZV TK, tissue–specific anabolism of araM to araATP, and tissue–specific cytotoxicity due to araM exposure. By utilizing such retroviral vectors, araM was anabolized to araATP in hepatoma cells, producing a selective cytotoxic effect.

Retroviral-mediated gene therapy for the treatment of hepatocellular carcinoma: an innovative approach for cancer therapy.

An approach involving retroviral-mediated gene therapy for the treatment of neoplastic disease is described. This therapeutic approach is called "virus-directed enzyme/prodrug therapy" (VDEPT). The VDEPT approach exploits the transcriptional differences between normal and neoplastic cells to achieve selective killing of neoplastic cells. We now describe development of the VDEPT approach for the treatment of hepatocellular carcinoma. Replication-defective, amphotrophic retroviruses were constructed containing a chimeric varicella-zoster virus thymidine kinase (VZV TK) gene that is transcriptionally regulated by either the hepatoma-associated alpha-fetoprotein or liver-associated albumin transcriptional regulatory sequences. Subsequent to retroviral infection, expression of VZV TK was limited to either alpha-fetoprotein- or albumin-positive cells, respectively. VZV TK metabolically activated the nontoxic prodrug 6-methoxypurine arabinonucleoside (araM), ultimately leading to the formation of the cytotoxic anabolite adenine arabinonucleoside triphosphate (araATP). Cells that selectively expressed VZV TK became selectively sensitive to araM due to the VZV TK-dependent anabolism of araM to araATP. Hence, these retroviral-delivered chimeric genes generated tissue-specific expression of VZV TK, tissue-specific anabolism of araM to araATP, and tissue-specific cytotoxicity due to araM exposure. By utilizing such retroviral vectors, araM was anabolized to araATP in hepatoma cells, producing a selective cytotoxic effect.

Selective induction of toxicity to human cells expressing human immunodeficiency virus type 1 Tat by a conditionally cytotoxic adenovirus vector.

The human immunodeficiency viruses (HIVs) primarily infect CD4+ T lymphocytes, leading eventually to the development of a systemic immune dysfunction termed acquired immunodeficiency syndrome (AIDS). An attractive strategy to combat HIV-mediated pathogenesis would be to eliminate the initial pool of infected cells and thus prevent disease progression. We have engineered a replication-defective, conditionally cytotoxic adenovirus vector, Ad-tk, whose action is dependent on the targeted expression of the herpes simplex virus type 1 thymidine kinase gene (tk), cloned downstream of the HIV-1 long terminal repeat, in human cells expressing the HIV-1 transcriptional activator Tat. Infection of Tat-expressing human HeLa or Jurkat cells with Ad-tk resulted in high-level tk expression, which was not deleterious to the viability of these cells. However, in the presence of the antiherpetic nucleoside analog ganciclovir, Ad-tk infection resulted in a massive reduction in the viability of these Tat-expressing cell lines. As adenoviruses are natural passengers of the human lymphoid system, our results suggest adenovirus vector-based strategies for the targeted expression, under the control of cis-responsive HIV regulatory elements, of cytotoxic agents in HIV-infected cells for the therapy of HIV-mediated pathogenesis.

Evidence for an involvement of thymidine kinase in the excision repair of ultraviolet‐irradiated herpes simplex virus in human cells

A wild-type strain of herpes simplex virus type 1 (HSV-1:KOS) encoding a functional thymidine kinase (tk+) and a tk- mutant strain (HSV-1:PTK3B) were used to study the role of the viral tk in the repair of UV-irradiated HSV-1 in human cells. UV survival of HSV-1:PTK3B was substantially reduced compared with that of HSV-1:KOS when infecting normal human cells. In contrast, the UV survival of HSV-1:PTK3B was similar to that of HSV-1:KOS when infecting excision repair-deficient cells from a xeroderma pigmentosum patient from complementation group A. These results suggest that the repair of UV-irradiated HSV-1 in human cells depends, in part at least, on expression of the viral tk and that the repair process influenced by tk activity is excision repair or a process dependent on excision repair.

Examination of the roles of transcription factor Sp1-binding sites and an octamer motif in trans induction of the herpes simplex virus thymidine kinase gene

Herpes simplex virus mutants with both Sp1-binding sites in the thymidine kinase (tk) promoter inactivated or an octamer motif deleted were at most modestly impaired for tk expression. Thus, no cellular transcription factor that binds upstream of the tk TATA box is solely required for trans induction of this gene.

Amplification of plasmid copy number by thymidine kinase expression in Saccharomyces cerevisiae.

A 2 micron circle-based chimaeric plasmid containing the yeast LEU2 and the Herpes Simplex Virus type 1 thymidine kinase (HSV-1 TK) genes was constructed. Transformants grown under selective conditions for the LEU2 gene harboured the plasmid at about 15 copies per cell whilst selection for the HSV-1 TK gene led to an increase to about 100 copies per cell. Furthermore, the plasmid copy number could be controlled by the stringency of selection for the TK gene, and the increase in TK gene dosage was reflected in an increase in intracellular thymidine kinase activity. The mitotic stability of the plasmid in "high-copy" and "low-copy" number cells was determined. "High-copy" number cells showed a greater mitotic stability. The relationship of TK expression to plasmid copy number may be useful for the isolation of plasmid copy number mutants in yeast and the control of heterologous gene expression.

A genetic approach to promoter recognition during trans induction of viral gene expression.

Viral infection of mammalian cells entails the regulated induction of viral gene expression. The induction of many viral genes, including the herpes simplex virus gene encoding thymidine kinase (tk), depends on viral regulatory proteins that act in trans. Because recognition of the tk promoter by cellular transcription factors is well understood, its trans induction by viral regulatory proteins may serve as a useful model for the regulation of eukaryotic gene expression. A comprehensive set of mutations was therefore introduced into the chromosome of herpes simplex virus at the tk promoter to directly analyze the effects of promoter mutations on tk transcription. The promoter domains required for efficient tk expression under conditions of trans induction corresponded to those important for recognition by cellular transcription factors. Thus, trans induction of tk expression may be catalyzed initially by the interaction of viral regulatory proteins with cellular transcription factors.

Trans Activation of the simian virus 40 enhancer.

We describe experiments which demonstrated that the simian virus 40 (SV40) enhancer affects certain transcriptional units differently. We also found that a specific enhancer-transcriptional unit interaction can be regulated by trans-acting factors. Using transient assays, we examined the effects of the SV40 enhancer on herpesvirus thymidine kinase (tk) RNA levels when transcription was initiated either by the herpesvirus tk promoter or by an SV40 early promoter-tk fusion. We were unable to detect any effect of the enhancer on transcription from the tk promoter in CV-1 or HeLa cells. However, we found that the addition of T-antigen in trans allowed the enhancer to stimulate expression from the tk promoter. This induction by T-antigen did not require T-antigen-binding sites in cis and appeared to be an indirect effect. In contrast, tk expression from the SV40 early promoter fusion was greatly stimulated by the enhancer in CV-1 cells. Furthermore, in 293 cells the SV40 enhancer had only a marginal effect on the SV40 promoter-tk fusion, whereas it strongly stimulated tk expression from the tk promoter. Our results raise the possibility that the enhancer function may not show cell specificity per se; rather, the interaction between the enhancer and a specific gene may be responsible for cell specificity. We discuss these observations in terms of the SV40 early gene-to-late gene switch that occurs during SV40 lytic growth.

Trans Activation of the simian virus 40 enhancer.

We describe experiments which demonstrated that the simian virus 40 (SV40) enhancer affects certain transcriptional units differently. We also found that a specific enhancer-transcriptional unit interaction can be regulated by trans-acting factors. Using transient assays, we examined the effects of the SV40 enhancer on herpesvirus thymidine kinase (tk) RNA levels when transcription was initiated either by the herpesvirus tk promoter or by an SV40 early promoter-tk fusion. We were unable to detect any effect of the enhancer on transcription from the tk promoter in CV-1 or HeLa cells. However, we found that the addition of T-antigen in trans allowed the enhancer to stimulate expression from the tk promoter. This induction by T-antigen did not require T-antigen-binding sites in cis and appeared to be an indirect effect. In contrast, tk expression from the SV40 early promoter fusion was greatly stimulated by the enhancer in CV-1 cells. Furthermore, in 293 cells the SV40 enhancer had only a marginal effect on the SV40 promoter-tk fusion, whereas it strongly stimulated tk expression from the tk promoter. Our results raise the possibility that the enhancer function may not show cell specificity per se; rather, the interaction between the enhancer and a specific gene may be responsible for cell specificity. We discuss these observations in terms of the SV40 early gene-to-late gene switch that occurs during SV40 lytic growth.

Effects of 20-OH-ecdysone on Drosophila cells : Regulation of endogenous and transfected genes

Drosophila cell lines respond to physiological doses of 20-OH-ecdysone by entering mitotic arrest and differentiating morphologically. The cells also exhibit changes in gene expression. Several enzyme activities are induced, and the synthesis of cytoplasmic actin and of the four small heat-shock proteins ( hsp) is initiated. Hybrid genes, containing the 5′ region of Drosophila heat-shock protein genes ligated to the herpes simplex virus thymidine kinase gene ( tk), have been transfected into cells of the Drosophila cell line S3. Constructions containing sequences upstream from hsp 70, or from any of the small hsp genes, show heat-inducible tk expression. Ecdysterone-inducible tk expression is seen only in transfections with small hsp-tk hybrid genes. This transient expression system can be used as an assay for function to define regions of DNA, flanking the coding region of inducible genes, which are necessary for normal gene expression and gene regulation in cultured cells.

Development of a helper-independent human adenovirus vector and its use in the transfer of the herpes simplex virus thymidine kinase gene.

Approximately 2 kilobases (kb) of additional DNA can be packaged into wild-type virions of human adenovirus type 5 (Ad5). To extend this limit, a helper independent Ad5 cloning vector was constructed by deleting most of early region 3 (E3) from map coordinates 78.5 to 84.7 and essentially all of early region 1 (E1) from coordinates 1.0 to 10.6. E3 is nonessential for adenovirus replication in cultured cells, and E1 is nonessential when the virus is propagated in 293 cells which constitutively express the E1 gene products. The resulting new virus, dlE1,3 is about 5.5 kb shorter than wild-type Ad5 and therefore should be able to accept up to 7.5 kb in foreign DNA. To test the usefulness of this vector, the herpes simplex virus type 1 (HSV-1) thymidine kinase gene (tk) along with its regulatory sequences was inserted into the unique XbaI site of dlE1,3 (at map position 78.5/84.7). The resulting recombinant virus, Adtk, expressed the HSV tk at a low level (as compared with HSV-1) in infected cells; however, tk expression was markedly enhanced when Adtk-infected cells were superinfected with a tk- mutant of HSV. Furthermore, the Adtk virus efficiently transformed tk- mouse cells (line LTA) to the tk+ phenotype. At a low efficiency, it was also possible to transform tk- human cells (line 143), and tk+ transformants of both mouse and human origin have been established as permanent lines.

Transcription of Drosophila small hsp-tk hybrid genes is induced by heat shock and by ecdysterone in transfected Drosophila cells.

Hybrid genes containing the 5' region of Drosophila heat shock protein (hsp) genes, ligated to the herpesvirus thymidine kinase (tk) gene, were transfected into Drosophila line S3 cells. Constructions containing sequences upstream from hsp70, or from any of the small hsp genes, showed heat-inducible tk expression. Ecdysterone-inducible tk expression was seen only in transfections with small hsp-tk hybrid genes. Plasmids containing a synthetic "heat shock consensus sequence" or a deletion of the hsp23 upstream region were totally inactive in transfection studies.

Promoter domains required for expression of plasmid-borne copies of the herpes simplex virus thymidine kinase gene in virus-infected mouse fibroblasts and microinjected frog oocytes.

A transient expression assay was used to measure the relative template activities of mutated tk genes in mouse L cells induced in trans by herpes simplex virus (HSV). In this assay, expression of the wild-type HSV type 1 tk gene is induced at least 200-fold by the superinfecting virus. Genetic lesions that were assayed include 5' deletions, clustered base substitutions, single base substitutions, intrapromoter inversions, and intrapromoter recombinants with the HSV type 2 tk gene. Roughly half of the mutations that were tested were found to weaken tk expression efficiency, and the remaining mutations did not alter expression. The spatial distribution of mutations that reduce expression efficiency in trans-induced mouse fibroblasts facilitated the construction of a map of promoter domains. The most gene-proximal promoter domain is located between 16 and 32 base pairs (bp) upstream of the tk mRNA cap site and contains a TATA homology. Two more distally located promoter domains were mapped to discrete locations upstream from the TATA homology. One of these distal domains is located between 47 and 79 bp upstream from the mRNA cap site, and the other is located between 84 and 105 bp upstream from the tk gene. The boundaries of these three promoter domains, with one exception, coincided with the set of domains delineated previously in a frog oocyte microinjection assay. The concordant behavior of tk promoter mutants in microinjected frog oocytes and trans-induced mouse fibroblasts leads us to propose that recognition and activation of the HSV tk promoter is mediated by cellular transcription factors that are common to frogs and mice.

Inhibition of HSV-transformed murine cells by nucleoside analogs, 2′-NDG and 2′-nor-cGMP: Mechanisms of inhibition and reversal by exogenous Nucleosides

A murine cell line transformed with HSV TK (LH-1) exhibits a greatly enhanced cytotoxicity to the nucleoside analog 9-[(2-hydroxy-1-(hydroxymethyl)ethoxy) methyl]guanine (2′-NDG) as compared to the parental LM cell line ( I 50 LH-1 = 0.4 μ M, I 50 LM = 44.4 μ M). Toxicity of 2′-NDG for LH-1 and LM is reversed only by the addition of 100 μ M thymidine (dThd), indicating that 2′-NDG is a substrate for the viral and cellular TK. In LM(TK −) cells-murine cells expressing no TK activity, 2′-NDG cytotoxicity is partially reversed only with dGuo. A cyclic phosphate derivative of 2′-NDG, 2′-norcGMP, contains a phosphodiester bond, is also taken up by cells, and does not depend on viral TK for activation. LH-1 cells and LM(TK −) cells are inhibited by similar concentrations of this analog (5.1 and 4.1 μ M, respectively). In all three cell lines (LM, LH-1, LM(TK-)), the toxicity of 2′-nor-cGMP is significantly reversed with dGuo or cyclic dGMP. This pattern of reversal differs significantly from that observed with 2′-NDG, suggesting that 2′-nor-cGMP is metabolized as a guanosine analog, similar to acyclovir, in LM and LM(TK −) cells. These results indicate that a cyclic monophosphate analog of 2′-NDG can be activated independently of viral TK expression and that cellular metabolic pathways resulting in elevated dGTP concentrations are important for reversal of toxicity induced by guanosine-like nucleoside analogs.

Promoter domains required for expression of plasmid-borne copies of the herpes simplex virus thymidine kinase gene in virus-infected mouse fibroblasts and microinjected frog oocytes.

A transient expression assay was used to measure the relative template activities of mutated tk genes in mouse L cells induced in trans by herpes simplex virus (HSV). In this assay, expression of the wild-type HSV type 1 tk gene is induced at least 200-fold by the superinfecting virus. Genetic lesions that were assayed include 5' deletions, clustered base substitutions, single base substitutions, intrapromoter inversions, and intrapromoter recombinants with the HSV type 2 tk gene. Roughly half of the mutations that were tested were found to weaken tk expression efficiency, and the remaining mutations did not alter expression. The spatial distribution of mutations that reduce expression efficiency in trans-induced mouse fibroblasts facilitated the construction of a map of promoter domains. The most gene-proximal promoter domain is located between 16 and 32 base pairs (bp) upstream of the tk mRNA cap site and contains a TATA homology. Two more distally located promoter domains were mapped to discrete locations upstream from the TATA homology. One of these distal domains is located between 47 and 79 bp upstream from the mRNA cap site, and the other is located between 84 and 105 bp upstream from the tk gene. The boundaries of these three promoter domains, with one exception, coincided with the set of domains delineated previously in a frog oocyte microinjection assay. The concordant behavior of tk promoter mutants in microinjected frog oocytes and trans-induced mouse fibroblasts leads us to propose that recognition and activation of the HSV tk promoter is mediated by cellular transcription factors that are common to frogs and mice.

Retrovirus transduction: segregation of the viral transforming function and the herpes simplex virus tk gene in infectious Friend spleen focus-forming virus thymidine kinase vectors.

A series of deletions and insertions utilizing the herpesvirus thymidine kinase gene (tk) were constructed in the murine retrovirus Friend spleen focus-forming virus (SFFV). In all cases, the coding region for the SFFV-specific glycoprotein (gp55), which is implicated in erythroleukemic transformation, was left intact. These SFFV-TK and SFFV deletion vectors were analyzed for expression of tk and gp55 after DNA-mediated gene transfer. In addition, virus rescued by cotransfection of these vectors with Moloney murine leukemia virus was analyzed for infectious TK-transducing virus, gp55 expression, and erythroleukemia-inducing ability. The experiments demonstrated that deletions or insertions within the intron for the gp55 env gene can interfere with expression of gp55 after both DNA-mediated gene transfer and virus infection. In contrast, the gene transfer efficiency of the tk gene was unaffected in the SFFV-TK vectors, and high-titer infectious TK virus could be recovered. Revertant viruses capable of inducing erythroleukemia and expressing gp55 were generated after cotransfection of the SFFV-TK vectors with murine leukemia virus. The revertant viruses lost both tk sequences and the ability to transduce TK- fibroblasts to a TK+ phenotype. These experiments demonstrate that segregation of the TK and erythroleukemia functions can occur in retrovirus vectors which initially carry both markers.

Retrovirus transduction: segregation of the viral transforming function and the herpes simplex virus tk gene in infectious Friend spleen focus-forming virus thymidine kinase vectors.

A series of deletions and insertions utilizing the herpesvirus thymidine kinase gene (tk) were constructed in the murine retrovirus Friend spleen focus-forming virus (SFFV). In all cases, the coding region for the SFFV-specific glycoprotein (gp55), which is implicated in erythroleukemic transformation, was left intact. These SFFV-TK and SFFV deletion vectors were analyzed for expression of tk and gp55 after DNA-mediated gene transfer. In addition, virus rescued by cotransfection of these vectors with Moloney murine leukemia virus was analyzed for infectious TK-transducing virus, gp55 expression, and erythroleukemia-inducing ability. The experiments demonstrated that deletions or insertions within the intron for the gp55 env gene can interfere with expression of gp55 after both DNA-mediated gene transfer and virus infection. In contrast, the gene transfer efficiency of the tk gene was unaffected in the SFFV-TK vectors, and high-titer infectious TK virus could be recovered. Revertant viruses capable of inducing erythroleukemia and expressing gp55 were generated after cotransfection of the SFFV-TK vectors with murine leukemia virus. The revertant viruses lost both tk sequences and the ability to transduce TK- fibroblasts to a TK+ phenotype. These experiments demonstrate that segregation of the TK and erythroleukemia functions can occur in retrovirus vectors which initially carry both markers.

Genetic effects of chromosomal rearrangements in Chinese hamster ovary cells: expression and chromosomal assignment of TK, GALK, ACP1, ADA, and ITPA loci.

Polyethylene glycol-mediated fusion of Chinese hamster ovary (CHO) cells with mouse Cl1D cells produced interspecific somatic cell hybrids which slowly segregated CHO chromosomes. Cytogenetic and isozyme analysis of HAT- and bromodeoxyuridine-selected hybrid subclones and of members of a hybrid clone panel retaining different combinations of CHO chromosomes enabled provisional assignments of the following enzyme loci to CHO chromosomes: TK, GALK, and ACP1 to chromosome 7; TK and GALK to chromosome Z13; ACP1, ADA, and ITPA to chromosome Z8; and ADA and ITPA to chromosome Z9. These genetic markers reflect the origin of each of these Z group chromosomes and indicate the functional activity of alleles located on rearranged chromosomes. Identification of diploid electrophoretic shift mutations for ADA and ITPA was consistent with those observations. Assignment of the functional TK locus in TK+/- CHO-AT3-2 cells indicated that gene deletion may be responsible for TK hemizygosity in this subline.

Plasmids expressing the Herpes simplex virus thymidine kinase gene in mammalian and bacterial cells.

Two plasmids containing either the complete thymidine kinase gene of Herpes simplex virus type I (pSK2) or the gene without the remote control sequence (pSK1) just behind the lac promoter and the first codons of the lacZ gene were constructed. Both plasmids efficiently transform mouse Ltk- cells as well as E. coli tk- cells to the Tk+ phenotype and are well suited for plasmid rescue from transformed mouse cells by direct functional selection for tk expression using a tk- mutant of E. coli C600.

Retrovirus long terminal repeats activate expression of coding sequences for the herpes simplex virus thymidine kinase gene.

In this study, we demonstrate that the long terminal repeats (LTRs) of a murine retrovirus can activate expression of heterologous gene coding sequences from which a functional promoter region has been deleted. Recombinant plasmid clones were obtained that contained both cloned fragments of Friend spleen locus-forming virus (SFFV) DNA and the herpes simplex virus (HSV) thymidine kinase (TK; ATP:thymidine 5'-phosphotransferase, EC 2.7.1.21) gene (tk). The effects of the LTR on tk expression were determined by constructing clones containing tk coding sequences with or without 5' sequences necessary for the initiation of transcription, inserted either 200 or 1200 base pairs downstream from the SFFV 5' LTR. The expression of the HSV TK protein by these clones was tested by gene transfer of the cloned into TK- mouse cells and assay of TK enzyme activity in TK+ transformants. These experiments demonstrate that: (i) the SFFV 5' LTR activates expression of tk coding sequences when these sequences are inserted 200 base pairs downstream from, and in the same orientation as, the LTR; (ii) tk is not activated when placed 1200 base pairs downstream from, and in the same orientation as, the LTR or when tk is inserted in either site in the opposite orientation as the LTR; (iii) the SFFV 5' LTR does not interfere with in vivo expression of tk when it is flanked by homologous 5' promoter sequences. The implication of these observations for retrovirus oncogenesis and animal cell genetics is discussed.