Phenotypic switching in cells transformed with the herpes simplex virus thymidine kinase gene

Biochemical transformation of Ltk- cells with the herpes simplex virus thymidine kinase (tk) gene resulted in numerous TK+ colonies that survived selection in hypoxanthine-aminopterin-thymidine medium. Many of these TK+ cell lines switched phenotypes and reverted to the TK- state. In this report, we describe the biological and biochemical characteristics of three TK- revertant lines. One (K1B5) transiently expressed TK in the presence of bromodeoxyuridine, which selects for the TK- phenotype. Another TK- sibling (K1B6n) expressed TK only after removal from bromodeoxyuridine-containing medium. The last variant (K1B6me) lost the ability to switch to the TK+ phenotype, although it maintained the herpes simplex virus sequences coding for TK. Loss of the ability of K1B6me cells to express TK was correlated with extensive methylation of the sequence recognized by the restriction endonuclease HpaII (pCpCpGpG). After these cells were treated with 5-azacytidine, they regained the ability to clone in hypoxanthine-aminopterin-thymidine medium and reexpressed virus tk mRNA and enzyme. In addition, the HpaII sites that were previously shown to be refractile to enzyme digestion were converted to a sensitive state, demonstrating that they were no longer methylated.     

Measuring herpes simplex virus thymidine kinase reporter gene expression in vitro

The herpes simplex 1 virus thymidine kinase (HSV1-tk) positron emission tomography (PET) reporter gene (PRG) or its mutant HSV1-sr39tk are used to investigate intracellular molecular events in cultured cells and for imaging intracellular molecular events and cell trafficking in living subjects. Two in vitro methods are available to assay gene expression of HSV1-tk or HSV1-sr39tk in cells or tissues. One method determines the level of HSV1-TK or HSV1-sr39TK enzyme activity in cell or tissue lysates by measuring the amount of the radiolabeled substrates that have been phosphorylated by these enzymes in a fixed amount of cell lysate protein after a fixed incubation time. The other method, called the 'cell-uptake assay', takes into account the natural uptake and efflux characteristics of the radiolabeled substrate by specific cells, in addition to the level of HSV1-TK or HSV1-sr39TK activity. Both of these assays can be used to validate molecular models in cultured cells, prior to studying them in living research subjects. Each of these assays can be completed in one day.     

3'-Amino thymidine affinity matrix for the purification of herpes simplex virus thymidine kinase.

A simple procedure for preparation of an affinity resin with 3''-amino thymidine linked to the carboxyl residues on 6-amino-hexanoic agarose is described. We have used this column for a rapid and simple purification of the thymidine kinase encoded by the herpes simplex virus type 1 genome. This resin has two major advantages over the most widely use used resin made with thymidine-p-nitrophenyl phosphate: first it is easily obtainable, and second, it is not subject to destruction by phosphodiesterases. The two resins are very similar in behavior and the resin made with amino thymidine has allowed us to prepare large quantities of highly purified HSV TK for crystallization studies.     

Nucleotide sequence of the herpes simplex virus type 2 thymidine kinase gene.

We have determined the complete nucleotide sequence of the thymidine kinase gene of herpes simplex virus (HSV) type 2 strain 333. The sequence of the thymidine kinase gene exhibits an open translational reading frame of 1,128 nucleotides encoding a protein of 376 amino acids. The DNA sequence was compared with that of the HSV type 1 thymidine kinase gene from strain MP (S. L. McKnight, Nucleic Acids Res. 8:5949-5964, 1980) and from strain CL 101 (M. J. Wagner, J. A. Sharp, and W. C. Summers, Proc. Natl. Acad. Sci. U.S.A. 78:1441-1445, 1981) to assess the extent of intra- and intertypic variation for one viral gene. The nucleotides encoding the structural gene varied 1.7% between the two HSV type 1 strains and 19% between HSV type 1 and HSV type 2, which translated to differences in the amino acid sequence of the two proteins of 1.9 and 27%, respectively. The DNA encoding the 5' regulatory sequences appeared to be more conserved than the DNA coding for the structural gene, and the DNA at the 3' end of the gene was the least homologous.     

Retrovirus—mediated herpes simplex virus thymidine kinase gene therapy approach for hepatocellular carcinoma

INTRODUCTI0NHepatocellularcarcinoma(HCC)is0ne0fthem0stc0mm0nhumanmalignancies,causinganestimatedl,250,OOOdeatht0llperyearworldwide[1].Thep0orprognosisencounteredintreatment0fsuchcarcinomaismainlycausedbylatediagn0sisandinsufficiency0feffectivestrategies,especiallyforadvanced-stagedpatients.However,recentknowledge0fpathogenesisofHCCatm0lecularlevelprovidesanalternativeappr0achwhenc0nsideringgenetherapyastreatmelltf0rHCC.Am0ngthevari0usgenetherapystrategiesincancer)itwaJsrep0rtedthatth…     

Initiation of herpes simplex virus thymidine kinase polypeptides.

When employed as a transgene reporter, the herpes simplex type 1 virus (HSV1) thymidine kinase gene (tk) is ectopically expressed in mouse testis. The principal testicular mRNA lacks the 5'-end of the tk reading frame. As a result the principal translation products, P2 and P3, are N-terminally truncated. These co-migrate in SDS-PAGE with polypeptides synthesised during HSV1 infection that were previously thought to be initiated at methionine codons ATG46 and ATG60. Prompted by these observations we generated modified tk genes each carrying only one of the first three ATG codons. Transfected cells expressed both full-length enzyme (P1) and P2 when only ATG1 was unmodified, P2 and P3 when only ATG46 was unmodified or P2 and a fourth polypeptide (P4) when only ATG60 was unmodified. Our observations indicate that P3 is initiated at ATG46 rather than ATG60, while P2 is initiated at a non-ATG codon rather than ATG46 and P4 is initiated at ATG60. When either of two putative non-ATG initiation codons was modified P2 was no longer produced. Cells mainly expressing either P1 or P3 exhibited the same sensitivity to Ganciclovir as cells transfected with the unaltered tk gene. P1 and P3 both have TK activity while P4 probably has none.     

The herpes simplex virus thymidine kinase gene is not transcribed in Saccharomyces cerevisiae

The herpes simplex virus thymidine kinase gene has been cloned into a chimeric yeast plasmid cloning vehicle and transformed into appropriate yeast strains. Plasmids carrying the herpes simplex virus thymidine kinase gene can be propagated as autonomously replicating plasmids, but no RNA specific to the thymidine kinase coding sequence was detected.     

Control of the expression of a herpes simplex virus thymidine kinase gene incorporated into thymidine kinase-deficient mouse cells.

When thymidine kinase-deficient mouse cells "transformed" by in activated herpes simplex virus and expressing the viral thymidine kinase (TK) are grown in nonselective medium, there is an exponential decay in the proportion of cells that continue to express the viral enzyme. However, the viral TK can be reactivated at a frequency of approximately 1 cell in 10(6) in every population that has lost TK activity. When cells in which the viral TK has been reactivated are grown in nonselective medium, a decay in the expression of the viral enzyme occurs again at the same rate as in the initial transformed population. Studies on the reactivation of viral TK indicate that reappearance of the enzyme is not induced by the selective medium (HAT) used to detect cells in which the enzyme has reappeared. Furthermore, treatments known to induce latent viruses in other systems--eg, exposure of the cells to mutagens or cell fusion--do not affect the frequency with which viral TK is reactivated.     

Location and cloning of the herpes simplex virus type 2 thymidine kinase gene.

The herpes simplex virus type 2 thymidine kinase gene has been mapped to a position colinear with the herpes simplex virus type 1 thymidine kinase gene and cloned within a 4.0-kilobase fragment in pBR 322.     

The nucleotide sequence and transcript map of the herpes simplex virus thymidine kinase gene

This paper presents the nucleotide sequence of the Herpes Simplex Virus thymidine kinase (tk) gene. The position on the DNA sequence corresponding to the 5' and 3' termini of tk messenger RNA have been mapped. The mRNA termini are separated by slightly more than 1,300 nucleotides. The same 2,300 nucleotide segment of tk coding strand DNA is fully protected from S1 nuclease digestion when hybridized to tk mRNA. The location and size of the mRNA-coding segment corresponds to a region of the viral DNA that is essential for tk gene expression in microinjected frog oocytes. The nucleotide sequence of the HSV tk gene exhibits an open translational reading frame of 376 codons that extends from the methionine codon most proximal to the 5' terminus of tk mRNA to a UGA stop codon approximately 70 nucleotides from the poly-A addition site. The results of these experiments indicate that the tk gene is not interrupted by intervening DNA sequences, and that certain oligonucleotide sequences adjacent to the termini of the tk gene are homologous to similarly positioned sequences common to structural genes of eukaryotic cells.     

The herpes simplex virus thymidine kinase gene as a conditional negative-selection marker gene in Arabidopsis thaliana.

The human herpes simplex virus thymidine kinase type 1 gene (HSVtk) acts as a conditional lethal marker in mammalian cells. The HSVtk-encoded enzyme is able to phosphorylate certain nucleoside analogs (e.g. ganciclovir, an antiherpetic drug), thus converting them to toxic DNA replication inhibitors. The utility of HSVtk as a conditional negative-selection marker was explored in Arabidopsis thaliana (L.) Heynh. HSVtk was introduced into Arabidopsis by Agrobacterium-mediated transformation. Transgenic plants were morphologically indistinguishable from wild type and exhibited normal fertility. Ganciclovir at 10(-5) to 10(-4) M drastically reduced shoot regeneration on transgenic, HSVtk+ root explants or callus formation on HSVtk+ leaf explants but did not affect the wild-type cultures. There was a 35-fold reduction in shoot regeneration 8 d after transfer to shoot-induction medium. Negative selection against HSVtk activity along with kanamycin selection was also efficient in Agrobacterium-mediated gene transfer experiments. Shoot regeneration was 25 times lower on double-selective (ganciclovir plus kanamycin) plates than in the kanamycin control. This regeneration rate in double-selective plates is in the range of the frequency of shoots normally escaping kanamycin selection in Arabidopsis cultures.     

Cytotoxicities of cytosine deaminase and herpes simplex virus thymidine kinase genes in melanoma cells are not affected by the promoter strength

The right choice of regulatory elements (promoters and enhancers) is essential for the preparation within cancer cells of the therapeutic genetic constructs aimed at the gene-programmed enzymatic transformation of non-toxic prodrugs into toxins. It is generally accepted that the efficiency of gene therapy constructions is dependent, in particular, on the strength of promoters regulating the expression of therapeutic genes. Using melanoma-specific promoters and gene enhancers of human melanoma inhibitory activity and mouse tyrosinase and therapeutic genes, namely, HSVtk encoding herpes simplex virus thymidine kinase and FCU1 encoding cytosine deaminase/uracil phosphoribosyltransferase hybrid protein gene, we demonstrated that the promoter strength was not critical for the development of cytotoxic effects. The cytotoxic activity of these genes was shown to be influenced by the concentration of the prodrug added.     

Adaptation of a retrovirus as a eucaryotic vector transmitting the herpes simplex virus thymidine kinase gene.

We investigated the feasibility of using retroviruses as vectors for transferring DNA sequences into animal cells. The thymidine kinase (tk) gene of herpes simplex virus was chosen as a convenient model. The internal BamHI fragments of a DNA clone of Moloney leukemia virus (MLV) were replaced with a purified BamHI DNA segment containing the tk gene. Chimeric genomes were created carrying the tk insert in both orientations relative to the MLV sequence. Each was transfected into TK- cells along with MLV helper virus, and TK+ colonies were obtained by selection in the presence of hypoxanthine, aminopterin, and thymidine (HAT). Virus collected from TK+-transformed, MLV producer cells passed the TK+ phenotype to TK- cells. Nonproducer cells were isolated, and TK+ transducing virus was subsequently rescued from them. The chimeric virus showed single-hit kinetics in infections. Virion and cellular RNA and cellular DNA from infected cells were all shown to contain sequences which hybridized to both MLV- and tk-specific probes. The sizes of these sequences were consistent with those predicted for the chimeric virus. In all respects studied, the chimeric MLV-tk virus behaved like known replication-defective retroviruses. These experiments suggest great general applicability of retroviruses as eucaryotic vectors.     

Chicken ovalbumin gene fused to a herpes simplex virus alpha promoter and linked to a thymidine kinase gene is regulated like a viral gene.

We are describing a system for the introduction, selection, and expression of eucaryotic genes in higher eucaryotic cells. The carrier consisted of the herpes simplex virus 1 (HSV-1) tk gene covalently linked to an HSV-1 alpha promoter directed away from the tk gene. In this study we fused to the alpha promoter the 5' transcribed noncoding sequences and the coding sequences of the chicken oviduct ovalbumin gene. Cells converted to the TK+ phenotype with this chimeric fragment produced an ovalbumin precursor which was processed and secreted into the extracellular fluid. The ovalbumin gene utilized the HSV-1 alpha promoter and was regulated as a viral gene inasmuch as inversion of the genomic DNA relative to the alpha promoter resulted in no ovalbumin synthesis, and production of ovalbumin was enhanced after superinfection with HSV-1. Synthesis of ovalbumin was not detected when cDNA was linked to the HSV-1 alpha promoter. The carrier system described in this study is suitable for introduction, selection, and expression of eucaryotic genes whose natural promoter is either weak or requires the presence of regulatory elements which may be absent from undifferentiated cells in culture.     

Interaction of cell and virus proteins with DNA sequences encompassing the promoter/regulatory and leader regions of the herpes simplex virus thymidine kinase gene

During the course of a productive infection with herpes simplex virus (HSV), gene expression is coordinately regulated in a cascade fashion. Three major kinetic classes of genes, termed alpha, beta, and gamma, are sequentially activated. The mechanism responsible for repression and subsequent activation of beta and gamma genes is not known. A mobility-shift electrophoresis assay was used to examine DNA fragments containing the promoter/regulatory and the mRNA leader regions of the thymidine kinase gene (TK, a model beta gene) for their ability to bind proteins present in nuclear extracts prepared from uninfected and infected cells. Specific complexes unique to each extract were formed. Using a monoclonal antibody specific for ICP4 (the major regulatory protein of HSV) we demonstrated that this protein is present in the complexes formed between probes encompassing either the promoter/regulatory or leader sequence DNAs and proteins in infected-cell extracts. These complexes formed despite the lack of a high affinity binding site for ICP4 in either of these regions. The stability of complexes formed in infected-cell extracts with DNA probes containing the promoter/regulatory, leader region, and a high affinity ICP4-binding site were compared by dissociation analysis. The relative kd(obs) for these DNA-protein complexes was in the order: TK-leader region much greater than TK-promoter/regulatory region greater than or equal to high affinity ICP4-binding site. Cu+/1,10-phenanthroline footprinting revealed that infected-cell complexes which form on a probe containing a high affinity ICP4-binding site generate a protection pattern, whereas those formed on a probe containing the TK-leader sequence do not. In contrast, complexes formed with the latter probe in extracts from uninfected cells are kinetically stable and refractile to cleavage. A model for activation of the TK gene which incorporates these results is presented.     

Properties of cells carrying the herpes simplex virus type 2 thymidine kinase gene: mechanisms of reversion to a thymidine kinase-negative phenotype

We have isolated cells with a thymidine kinase-negative (tk⁻) phenotype from cells which carry the herpes simplex virus type 2 tk gene by selection in 5-bromodeoxyuridine or 9-(2-hydroxyethoxymethyl)guanine. Both selection routines generated revertants with a frequency of 10⁻³ to 10⁻⁴, and resistance to either compound conferred simultaneous resistance to the other. tk⁻ revertants fell into three classes: (i) cells that arose by deletion of all virus sequences, (ii) cells that had lost the virus tk gene but retained a nonselected virus-specific function and arose by deletion of part of the virus-specific sequence, and (iii) cells that retained the potential to express all of the virus-specific functions of the parental cells and retained all of the virus-specific DNA sequences.