Saul Kit

Mutant strains of herpes simplex deficient in thymidine kinase-inducing activity

Abstract A mouse fibroblast subline [strain LM (TK−)] which lacks thymidine (TdR) and deoxyuridine (UdR) kinase activities acquires these enzymes following infection by herpes simplex virus. Mutant strains of herpes simplex have been isolated, which are resistant to bromodeoxyuridine (BUdR) and iododeoxyuridine (IUdR), and deficient in enzyme-inducing activity. The herpes simplex mutants fall into 3 groups: (1) mutant strains that induce either very low levels of TdR kinase activity or none at all; (2) mutant strains that induce approximately normal levels of TdR-H3 uptake into the nuclei of 4–14% of the LM (TK−) cells; and (3) mutant strains that induce only very light TdR-H3 labeling of the nuclei of 22–63% of the LM (TK−) cells. Three mutants from group I have been propagated for several passages in LM or in LM (TK−) cells in media lacking BUdR. LM (TK−) cells were then challenged with virus from each of the passages and tested for TdR-H3 incorporating activity. The experiments demonstrated that these mutants do not undergo gross reversion. Infection with parental herpes simplex enhanced the TdR and UdR kinase activities of LM cells, while infection with herpes simplex mutants reduced these enzymatic activities. The uridine kinase activity of both LM and LM (TK−) cells was reduced following inoculation of cultures with either parental or mutant herpes simplex strains. Growth of parental herpes simplex in LM (TK−) cells was markedly inhibited by either 25 μg/ml BUdR or 25 μg/ml IUdR. Similarly growth of parental virus in LM cells was inhibited by 25 μg/ml BUdR. However, growth of mutant 2011 occurred in LM (TK−) cells in the presence of 500 μg/ml BUdR and was only partially inhibited in LM cells at a BUdR concentration of 200 μg/ml BUdR. Growth of mutant 2006 in LM (TK−) cells was not inhibited by 250 μg/ml IUdR.

EFFECT OF HALOGENATED PYRIMIDINES AND THYMIDINE ON GROWTH OF L-CELLS AND A SUBLINE LACKING THYMIDINE KINASE.

Summary The effects of bromodeoxyuridine (BUDr), iododeoxyuridine (IUDr), thymidine (TDr), fluorodeoxyuridine (FUDr), and fluorouracil (FU), on the growth of mouse fibroblasts (Strain L-M) and on a bromodeoxyuridine resistant subline [Stain L-M (TK−)] have been compared. The growth of L-M cells was grossly inhibited by 25 μg/ml BUDr and 50 μg/ml IUDr while L-M (TK−) cells grew for at least 5 generations in 1 mg/ml BUDr or 200 μg/ml IUDr. High concentrations (100 to 200 μg/ml) of TDr inhibited the growth of L-M cells, but L-M (TK−) cells were insensitive to these TDr concentrations. The L-M cells were approximately 250-fold more sensitive than L-M (TK−) cells to fluorodeoxyuridine (FUDr). The inhibitory effects of FUDr (0.01 μg/ml) on the growth of L-M cells was reversed by TDr (10 μg/ml), even when the TDr was added 24 hr after the addition of FUDr. However, TDr (100 μg/ml) did not prevent the inhibition by FUDr (2.5 μg/ml) of L-M (TK−) cell growth. The growth of either cell line was inhibited by 5-fluorouracil. The relationships of these results to the finding that thymidine kinase activity is deleted from L-M (TK−) cells are discussed.

Properties of deoxythymidine kinase partially purified from noninfected and virus-infected mouse fibroblast cells

Abstract Deoxythymidine kinase, partially purified from noninfected LM mouse fibroblast cells, was rapidly inactivated when incubated in the absence of substrates at 38°C. The half-life of the enzyme was 29–43 minutes whether prepared from LM cells in the logarithmic phase of growth, the phase of negative growth acceleration, the stationary phase, or from LM cells which had been incubated for 24 hours with 50 μg deoxythymidine per milliliter prior to harvest. The deoxythymidine kinase induced in LM (TK−) cells by herpes simplex virus was also very unstable. In contrast, deoxythymidine kinase prepared from vaccinia-infected LM or LM (TK−) cells was considerably more stable with a half-life greater than 300 minutes. The addition of ATP (15 mM), dTTP (0.025 mM), or deoxythymidine (0.05 to 0.1 mM) significantly protected the LM cell enzyme against thermal inactivation. However, charcoal treatment of the vaccinia-induced enzyme did not decrease the stability of that enzyme. Immunological differences and differences in apparent Michaelis constants for deoxyuridine were also observed between the vaccinia-induced deoxythymidine kinase and the enzyme from noninfected LM cells. However, the temperature characteristics of the reactions, calculated from the Vm values at 38° and 30°, were similar, and these enzymes were nearly equal in susceptibility to inhibition by dTTP. With either enzyme, the phosphorylation of deoxyuridine was competitively inhibited by fluorodeoxyuridine, bromodeoxyuridine, iododeoxyuridine, or deoxythymidine. The phosphorylation of deoxythymidine was inhibited by low concentrations of bromodeoxyuridine or iododeoxyuridine, and by high concentrations of deoxyuridine or fluorodeoxyuridine.

Enzyme induction in SV40-infected green monkey kidney cultures

Abstract After infection of primary cultures of green monkey kidney cells or of an established green monkey kidney cell line (CV-1) with SV40, thymidine kinase activity was induced. Increased enzyme activity was first detectable 12–16 hours after inoculation and was 4–15 times greater at 28–48 hours in infected than in noninfected cultures. Thymidine kinase has been partially purified from noninfected and SV40-infected cells. With deoxyuridine (UdR) as substrate, the Michaelis constant of the enzyme from infected cells was 2–3 times greater than that from noninfected cells. However, the kinetics of inactivation of the enzymes were about the same at 38 °. 1-β- d -Arabinofuranosylcytosine (ara-C) inhibited the incorporation of tritiated thymidine (H3-TdR) into the deoxyribonucleic acid (DNA) of noninfected or SV40-infected monkey kidney cultures by 95% or more. Ara-C treatment increased greatly the thymidine kinase activity of noninfected monkey kidney cultures. The Michaelis constant of thymidine kinase purified from ara-C treated CV-1 cultures was identical to that from untreated cultures. Ara-C did not prevent the induction of thymidine kinase by SV40. Indeed, the effects of ara-C and SV40 on enzyme induction were approximately additive in drug treated and infected cultures. Bromodeoxyuridine (BUdR) treatment did not prevent thymidine kinase induction by SV40, although virus growth was inhibited by 99.9%. Unlike ara-C, BUdR did not have a stimulating effect on thymidine kinase activity in noninfected cells. Cycloheximide inhibited the incorporation of radioactive amino acids into proteins of monkey kidney cells. Cycloheximide prevented thymidine kinase induction when added to cultures early after SV40 infection and arrested further enzyme induction when added after thymidine kinase induction had begun. A gradual twofold increase in thymidylate synthetase activity occurred in SV40-infected cultures but thymidine 5′-monophosphate (TMP) kinase and uridine kinase activities increased by less than 50%. Radioautographic and biochemical experiments on the incorporation of radioactive precursors into DNA demonstrated that the percentage of cells with labeled nuclei and the uptake of either tritiated deoxyadenosine or H3-TdR into DNA was sharply increased beginning at about 16–20 hours after infection. The total DNA content of SV40-infected cultures was also increased.

ISOLATION AND PROPERTIES OF VACCINIA MUTANTS DEFICIENT IN THYMIDINE KINASE-INDUCING ACTIVITY.

Abstract A bromodeoxyuridine (BUdR)-resistant mouse fibroblast subline [Strain LM (TK − )] lacks thymidine (TdR) and deoxyuridine (UdR) kinase activities. These enzyme activities are induced, however, following infection of the LM (TK − ) cells by parental vaccinia virus. Mutant vaccinia strains have been isolated which are deficient in TdR and UdR kinase-inducing activities. The mutant virus strains are relatively stable; they can be passed at least five times in LM or in LM (TK − ) cells in media lacking BUdR without gross reversions. Parental LM mouse fibroblast cells display TdR and UdR kinase activities, and the enzyme activities are increased following infection by parental vaccinia. However, after infection of LM cells with mutant vaccinia strains, the TdR and UdR kinase activities are decreased. Parental and mutant vaccinia strains reduce the uridine kinase activity and the incorporation of deoxycytidine-H 3 into the DNA of both LM and LM (TK − ) cells, and also reduce the incorporation of TdR-H 3 into the DNA of the parental LM cells. Growth of vaccinia mutant 1004 in LM (TK − ) cells is not inhibited by 200 μg BudR or IUdR per milliliter and is only slightly reduced by 500 μg BUdR per milliliter. BUdR concentrations of 50 μg/ml or higher are required to inhibit growth of mutant 1004 in LM cells. However, BUdR or IUdR concentrations of 25 μg/ml suppress the growth of parental vaccinia in LM (TK − ) cells, and BUdR at 25 μg/ml inhibits the growth of parental vaccinia in LM cells.

Effects of 5-fluorouracil, actinomycin D and mitomycin C on the induction of thymidine kinase by vaccinia-infected L-cells

The effects of actinomycin D, 5-fluorouracil, 5-fluorodeoxyuridine and mitomycin C on the induction of thymidine kinase in vaccinia-infected mouse fibroblasts were investigated. When cells were treated with high levels of actinomycin D 1 hr prior to the addition of vaccinia, thymidine kinase induction was prevented. However, when cells were pretreated for 8 hr with 1 μ g/ml. actinomycin D 24 hr before the inoculation of virus, thymidine kinase induction was reduced to only about half the control level, although the incorporation of [ 3 H]uridine into RNA was inhibited by about 95%. 5-Fluorouracil prevented the incorporation of [ 3 H]uridine into the DNA-thymine of both non-infected and vaccinia-infected cells. Either in the absence or in the presence of 5-fluorouracil or 5-fluorodeoxyuridine, there was no detectable incorporation of [ 3 H]thymidine into the DNA of non-infected L-M (TK−) cells. Following infection of L-M (TK−) cells with vaccinia, however, 5-fluorouracil and 5-fluorodeoxyuridine enhanced the incorporation of [ 3 H]thymidine into DNA. 5-Fluorouracil (100 to 300 μ g/ml.) caused only moderate reductions in the induction of thymidine kinase activity in vaccinia-infected cells. Antecedent treatment of non-infected L-cells for 1 hr with 50 μ g/ml. mitomycin C or for 17 hr with 15 μ g/ml. mitomycin C, respectively, reduced the incorporation for 1 hr of [ 3 H]thymidine into DNA by 95% and 99%. However, high levels of mitomycin C did not prevent thymidine kinase induction in vaccinia-infected cells. When L-M (TK−) cells were pretreated for 16 hr with 15 μ ,g/ml. mitomycin C, infected with vaccinia virus, and further treated with 48·5 μ g/ml. mitomycin C at 1 hr after inoculation, thymidine kinase induction was only reduced by about 46%. The results support the concept that vaccinia DNA-dependent RNA synthesis is needed for thymidine kinase induction, but that the suppression of DNA synthesis or host-cell DNA-dependent RNA synthesis does not prevent enzyme induction.

The effect of temperature on induction of deoxythymidine kinase activity by herpes simplex mutants

Abstract A series of bromodeoxyuridine (BUdR)-resistant vaccinia and herpes simplex mutants which exhibit defective deoxythymidine kinase-inducing activity have recently been isolated ( Dubbs and Kit, 1964 , Dubbs and Kit, 1964 ). Of the mutants, some, such as herpes simplex mutant 132006 and vaccinia mutants 131004, 111011, and 131015, induced extremely low levels of deoxythymidine kinase when infected cells were incubated at 37° or 31°. The replication of these mutants at the higher or the lower temperature was resistant to high levels of BUdR. Two of the herpes simplex mutants, i.e., 132010 and 132015, proved to be “leaky” mutants. Enzymatic, radioautographic, and antimetabolite experiments have shown that the induction of deoxythymidine kinase activity in LM (TK-) cells is a temperature-dependent process. When virus replication took place at 37°: (1) essentially no deoxythymidine kinase activity was detected in sonic extracts of infected cells when assayed at 38° or 30°; (2) approximately 15% of the cells at 2–10 hours post inoculation displayed more than 40 grain counts of TdR-H3 incorporation per nucleus; and (3) replication was not significantly inhibited by 100 μg/ml BUdR. However, when virus replication was studied at 31°: (1) approximately one-tenth the level of deoxythymidine kinase obtained with parental virus strains was induced; (2) about 50–75% of the cells had more than 40 grains per nucleus when the infected cells were incubated with TdR-Ha; and (3) replication of mutant 132010 was partly inhibited and that of 132015 almost completely inhibited by BUdR. Once induced at 31° by mutants 132010 and 131015, deoxythymidine kinase could be assayed at 38° or 30°. Growth of parental and mutant viral strains at 37° was inhibited by FUdR. TdR almost completely reversed the FUdR inhibition of the growth of parental viral strains and partially reversed that of herpes simplex mutants 132010 and 132015, but was less effective in reversing the FUdR inhibitions of the growth of herpes simplex mutant 132006 and vaccinia mutant 131004.

DNA polymerase induced by Simian virus 40.

Summary Following SV 40 infection of monkey kidney (CV-1) cells, DNA polymerase activity was induced. Enzyme formation was inhibited by cycloheximide, but not by 1-β-d-arabinofuranosylcytosine. Enhanced levels of DNA polymerase activity were also found in mouse kidney cell lines which had been transformed by SV 40. The DNA polymerases from uninfected and SV 40-infected CV-1 cells were partially purified and compared. Both enzymes required denatured DNA as primer and were similar with respect to pH optima, amount of primer required for maximal activity, kinetics of thermal denaturation, and sensitivity to inhibition by ammonium sulphate and sodium dodecyl sulphate. However, the Michaelis constant (Km ) for [3H]thymidine 5′-triphosphate of the SV 40-induced enzyme was about half that of the enzyme from uninfected cells. The Km value of DNA polymerase partially purified from SV 40-transformed mouse cells was about the same as that of the enzyme from uninfected CV-1 cells.

Induction of cytoplasmic ribonucleic acid (RNA) synthesis in vaccinia-infected LM cells during inhibition of protein synthesis☆

Abstract The effects of inhibitors of protein synthesis on the ribonucleic acid (RNA) synthesis of vaccinia-infected mouse fibroblast cells have been investigated. Cyclohexamide, puromycin, and dl - p -fluorophenylalanine (FPA) did not inhibit the induction of cytoplasmic RNA synthesis by vaccinia but did inhibit the incorporation of tritiated (H 3 ) leucine into protein and the induction by vaccinia of thymidine kinase. The results suggest that the RNA polymerase which catalyzes vaccinia-induced RNA synthesis preexists in the cell before infection or that it exists in the infecting virus particles. Joklik (1964) has shown that FPA and puromycin prevent the degradation of infecting vaccinia virions to a form in which the deoxyribonucleic acid (DNA) is susceptible to DNase digestion. Thus, from the findings presented here and those of Joklik (1964) , it is inferred that infecting vaccinia virions may stimulate RNA synthesis without becoming susceptible to DNase digestion. Vaccinia-induced RNA synthesis was less sensitive to inhibition by actinomycin D than was cellular RNA synthesis. Proflavine sulfate was shown to be a potent inhibitor of vaccinia-induced RNA synthesis and vaccinia-induced thymidine kinase formation.

Effects of age of culture and vaccinia infection on uridine kinase activity of L-cells☆

Abstract Sonic extracts of mouse fibroblast cells actively converted uridine to uridine nucleotides and degraded uridine to uracil. The uridine kinase, but not the uridine phosphorylase activity, varied considerably, depending on the time after subculture of the cells that the enzyme extracts were prepared. The highest levels of uridine kinase activity were observed at about 24 hr after subculture. An increase in enzymatic activity occurred during the lag phase preceding exponential growth. However, between 24 and 72 hr after subculture, the uridine kinase activity declined to about one-third the level at 24 hr. Following vaccinia infection, the uridine kinase activity of mouse fibroblast cells fell sharply. At about 7 hr post inoculation, the enzymatic activity of infected 2-day-old L-M cells decreased to about one third of the activity of uninfected cells. The fall of uridine kinase activity was more pronounced with vaccinia infected 2 and 3-day-old cells than with infected 1-day-old cells. The enzymatic activity of a mixture prepared by adding one volume of a sonic extract from vaccinia infected cells to one volume of a sonic extract from uninfected cells, was approximately equal to the sum of the activities of each of the respective sonicates, thus contraindicating the presence of potent enzyme inhibitors in the extracts from the infected cells. In contrast to the uridine kinase activity, uridine phosphorylase activity of 2-day-old mouse fibroblast cells was not significantly changed following vaccinia infection.