Site specificity of DNA methylation and expression of herpes simplex thymidine kinase gene.

Analysis of the methylation pattern of a single-copy herpes simplex virus thymidine kinase gene integrated into the genome of mouse L cells revealed that hypomethylation of five specific AvaI sites correlates with expression of the TK gene in all of the cell lines tested. Of these specific sites, one lies 5' to the coding region, one 3' to the coding region, and three lie within the coding region of the thymidine kinase gene. Analysis of methylation at a variety of other sites using other methylation-sensitive endonucleases revealed considerable variation in the methylation patterns, apparently unrelated to gene expression and subject to variation with time in culture.

Drosophila P element-enhanced transfection in mammalian cells.

We constructed a gene transfer vector containing the herpes simplex virus type 1 thymidine kinase (TK) gene flanked by Drosophila P element terminal repeats (W. R. Engels, Annu. Rev. Genet. 17:315-344). This vector was introduced into mouse LTK- cells and enhanced the frequency of stable transformation to the TK+ phenotype by approximately 50-fold relative to a similar plasmid lacking the P element terminal repeats.

Ultraviolet-induced reactivation, amplification, and hypomethylation of a herpes simplex virus thymidine kinase gene.

A line of mouse cells containing a methylated inactive herpes simplex virus thymidine kinase (TK) gene was irradiated with ultraviolet (UV) light in an attempt to induce expression of the inactive TK gene. UV irradiation was shown to be capable of inducing expression of the viral TK gene in a dose-dependent manner. Analysis of the methylation pattern of the viral TK gene indicated that the active TK gene in three UV-induced TK+ cell line was methylated to a lesser extent than was the inactive viral TK gene in the parental cells. Analysis of the copy number of the viral TK gene in parental and UV-induced cell lines also revealed that the viral TK gene was amplified 3- to 20-fold in three of four UV-induced TK+ cell lines tested.

Insulin-induced reactivation of an inactive herpes simplex thymidine kinase gene.

A line of mouse cells transformed with ultraviolet-irradiated herpes simplex virus type 1 and containing a methylated and inactive viral thymidine kinase (TK) gene was treated with insulin in an attempt to induce expression of the inactive gene. Insulin was found to be capable of inducing the inactive TK gene in these cells. The induction of the TK+ phenotype was dose dependent (from 1-100 micrograms of insulin per ml), and the TK activity induced was shown to be of viral origin. Analysis of the methylation pattern of the viral TK gene by using the methylation-sensitive restriction endonucleases Sma I, Hpa II, and Hha I revealed that the active viral TK gene in the parental transformed cells was hypomethylated, whereas the inactive TK gene in the uninduced TK- cells was methylated. The active TK gene in three insulin-induced TK+ lines also was methylated, but the methylation patterns in the insulin-induced lines all were different from the uninduced TK- line. These data suggest that extensive hypomethylation of the inactive TK gene is not required for insulin induction. Four other transformed lines containing an inactive viral TK gene were tested for insulin inducibility, but insulin was unable to induce expression of the TK gene in any of the other lines. Thus, insulin inducibility does not seem to be a function of the viral TK gene itself. These results suggest that insulin inducibility of the viral TK gene may be a reflection of the region of the host genome into which the TK gene was integrated.

5-Azacytidine-induced reactivation of a herpes simplex thymidine kinase gene.

Mouse cells transformed with herpes simplex virus and containing the viral thymidine kinase (TK) gene in an inactive state were treated with 5-azacytidine. The result was the reexpression of the viral TK gene. Two days of exposure to 5-azacytidine followed by 2 days of expression time was sufficient for maximal induction of the TK+ phenotype. The induction of TK expression by 5-azacytidine was concentration-dependent, with maximal induction at 10 micromoles per liter. 5-Azacytidine also inhibited the decay of TK expression in TK+ transformants removed from selective conditions. Analysis of the methylation patterns of the viral TK gene with restriction endonucleases Hpa II and Msp I showed the active gene to be unmethylated, the inactive gene methylated, and the 5-azacytidine-induced gene unmethylated.

Effects of nucleoside antivirals and their metabolites on the corneal endothelium.

The endothelial surface of rabbit corneas were perfused in vitro with bicarbonate Ringer's containing 5 mM glucose, 0.3 mM reduced glutathione, and various concentrations of nucleoside antivirals or their metabolites. During three hour perfusions, the swelling rates of corneas perfused with buffer containing either antivirals or metabolites were not significantly different from controls. Scanning electron microscopy of the endothelial cell layer revealed no structural abnormalities in any treatment group. One metabolite, fluoride ion, reduced endothelial glucose oxidation by about 60 percent when incubated with corneal tissue in vitro. The inhibition of glucose metabolism by fluoride ions was observed only at concentrations at least sixty times greater than would be anticipated in the anterior chamber of patients receiving topical F3TdR therapy. These studies indicate that 5-trifluoromethyl-2'-deoxyuridine, 5-iodo-2'-deoxyuridine, 9-(2-hydroxyethoxymethyl)-guanine, and their metabolites do not alter endothelial function when studied at physiological concentrations over a short term of exposure.

Biological effects of 5-carboxy-2'-deoxyuridine: hydrolysis product of 5-trifluoromethyl-2'-deoxyuridine.

5-Carboxy-2'-deoxyuridine (5-COOH-2'-dUrd) is a product of the base-catalyzed hydrolysis of 5-trifluoromethyl-2'-deoxyuridine. Hydrolysis of 5-trifluoromethyl-2'-deoxyuridine to 5-COOH-2'-dUrd in phosphate-buffered saline was kinetically first order and was pH dependent. At 37 degrees C and pH 7.0, 7.5, and 8.0, hydrolysis occurred with rate constants of 4.19 x 10(-5), 9.30 x 10(-5), and 1.61 x 10(-4) s(-1), respectively, with corresponding half-lives of 45.7, 20.6, and 11.9 h. 5-COOH-2'-dUrd inhibited growth of HEp-2 cells by 21, 67, and 91% at 1.0, 10, and 100 muM, with no antiviral activity against herpes simplex virus type 1 or herpes simplex virus type 2 at 1.0 or 10 muM. Partial reversal of cytotoxicity in HEp-2 cells was achieved with orotidine, uridine, deoxythymidine, or deoxycytidine, whereas complete reversal of cytotoxic effects was achieved with simultaneous addition of deoxythymidine, deoxycytidine, and uridine. 5-COOH-2'-dUrd at 50 muM inhibited incorporation of [(14)C]orotate into RNA and DNA by 65 and 27%, respectively. 5-COOH-2'-dUrd had no effect on the incorporation of [(3)H]uridine into DNA or RNA. Because of the structural similarities to deoxythymidine, 5-COOH-2'-dUrd was tested as an inhibitor of deoxythymidine kinase. 5-COOH-2'-dUrd was neither a substrate nor an inhibitor of herpes simplex virus type 1 induced deoxythymidine kinase or HEp-2 cell deoxythymidine kinase. Based on these observations, the metabolic block induced by 5-COOH-2'-dUrd has been localized to the de novo pyrimidine biosynthetic pathway between orotate phosphoribosyl transferase and orotidine 5'-phosphate decarboxylase.

Experimental herpes simplex virus type 1 encephalitis: treatment with 5-trifluoromethyl-2'-deoxyuridine.

5-Trifluoromethyl-2'-deoxyuridine (F(3)dThd) was evaluated for its neurotoxicity and for its ability to increase the life span of mice injected intracerebrally with herpes simplex virus type 1 (HSV-1) and F(3)dThd simultaneously. F(3)dThd showed no neurotoxicity at the highest concentration tested (100 mg/kg). Mice injected intracerebrally with HSV-1 died within 5 days postinfection. However, all mice injected concurrently with HSV-1 and 100 mg of F(3)dThd per kg lived through the termination of the experiment (60 days). Protection of mice from HSV-1 encephalitis by F(3)dThd has been shown to be dose dependent, with 100, 75, 50, and 25 mg of F(3)dThd per kg yielding a survival rate of 100, 90, 50, and 10%, respectively. HSV-1 titers in mouse brains receiving HSV-1 and 100 mg of F(3)dThd per kg concurrently were 100- to 1,000-fold lower at 2 to 4 days postinfection than control mice receiving HSV-1 alone. F(3)dThd was shown not to stimulate interferon production. Encephalitis caused by a ribonucleic acid virus, encephalomyocarditis virus, was not modified by F(3)dThd treatment.