Sensitivity to cell killing and the induction of cytogenetic damage following gamma irradiation in wild-type and thymidine kinase-deficient Friend mouse erythroleukaemia cells

Wild-type Friend erythroleukaemia (clone 707) cells and 2 thymidine kinase-deficient subclones, 707BUE and 707BUF, having thymidine kinase activities of 1.4% and 0.7% that of clone 707, were compared for sensitivity to killing and the induction of cytogenetic damage following irradiation. Three doses of gamma irradiation were used (150, 300 and 450 cGy), and cells were harvested for metaphase spreads after 4, 8, 12, 15, 29 and 43 h. G₂ delay was evident at 4 h following gamma irradiation in the 3 cell clones examined, and recovery of mitosis was observed to be dose-dependent. G₂ delay was found to be most prolonged in subclone 707BUE and most prompt in clone 707. Increased sensitivity to the induction of cytogenetic aberrations at all three doses was apparent in the 2 thymidine kinase-deficient subclones (as compared to wild-type cells) at 15, 29 and 43 h. Th thymidine kinase-deficient subclones also showed increased sensitivity to gamma radiation-induced cell killing. Furthermore, subclone 707BUE consistently exhibited greater to gamma irradiation than did the subclone with lower thymidine kinase activity, 707BUF. The importance of thymidine kinase levels and extended G₂ delay for DNA repair processes is discussed.     

A mammalian cell mutant with temperature-sensitive thymidine kinase.

We have isolated a mutant clone from mouse FM3A cells with temperature-sensitive defects both in cytokinesis and in thymidine kinase enzyme activity. The clone, designated tsCl.B59, was isolated after mutagenesis at 33 degrees C followed by exposure to cytosine arabinoside at 39 degrees C. It was derived from a thymidine kinase deficient, 5-bromodeoxyuridine-resistant clone (S-BUCl.42) which was originally derived from wild-type clone H-5 of FM3A cells. The temperature-sensitive mutant clone grows normally at 33 degrees C, but not at 39 degrees C, where it exhibits an increased frequency of multinucleate cells due to defective cytokinesis. Unlike the parental S-BUCl.42 cells, which have negligible thymidine kinase activity and are unable to incorporate 3H-thymidine, the mutant in corporates substantial amounts of 3H-thymidine at 33 degrees C, although its thymidine kinase activity remains lower than that of wild-type H-5 cells. When cultures of tsCl.B59 cells are transferred to 39 degrees C, incorporation of 3H-thymidine decreases markedly. The decrease has been shown to be due to thermolability of the thymidine kinase in tsCl.B59 cells.     

Protein kinase A (PKA) isoform RIIbeta mediates the synergistic killing effect of cAMP and glucocorticoid in acute lymphoblastic leukemia cells

Protein kinase A (PKA) or cAMP-dependent protein kinase (cAPK) mediates the synergistic effects of cAMP- and glucocorticoid (GC)-induced apoptosis in lymphoid cells. Using two human acute lymphoblastic leukemia cell (CEM) clones with respective GC-sensitive and GC-resistant phenotypes, we discovered that the PKA regulatory subunit isoform RII(beta) is preferentially expressed in the GC-sensitive clone C7-14 cells, whereas other intracellular cAMP receptors, including the exchange proteins directly activated by cAMP (Epac), are expressed at similar levels in both GC-sensitive and GC-resistant clones. High RII(beta) expression level in C7-14 cells is associated with elevated total PKA cellular activity and cAMP sensitivity, which consequently lead to an increased basal PKA activity. cAMP analogs that selectively activate type II PKA recapitulate the effects of forskolin of promoting apoptosis and antagonizing AKT/PKB activity in both GC-sensitive and GC-resistant clones, whereas type I PKA-selective agonists do not. Furthermore, down-regulation of RII(beta) leads to increased AKT/PKB activation and enhanced GC resistance in C7-14 cells. These results demonstrate that PKA RII(beta) is responsible for increased GC sensitivity, critical for cAMP-mediated synergistic cell killing in CEM cells, and may represent a novel therapeutic target for GC-resistant lymphoid malignancy.     

Thymidine Kinase 1 Deficient Cells Show Increased Survival Rate After UV-Induced DNA Damage

Balanced deoxynucleotide pools are known to be important for correct DNA repair, and deficiency for some of the central enzymes in deoxynucleotide metabolism can cause imbalanced pools, which in turn can lead to mutagenesis and cell death. Here we show that cells deficient for the thymidine salvage enzyme thymidine kinase 1 (TK1) are more resistant to UV-induced DNA damage than TK1 positive cells although they have thymidine triphosphate (dTTP) levels of only half the size of control cells. Our results suggest that higher thymidine levels in the TK- cells caused by defect thymidine salvage to dTTP protects against UV irradiation.     

Genetic transformation of somatic cells. IV. The rate of loss of transformant phenotype depends on the structure of the transforming DNA and changes when the cells are treated with the tumor promotor 12-o-tetradecanoyl-phorbol-13-acetate

Analysis was made of the phenotype stability of some clones of thymidine kinase deficient (TK-) Chinese hamster cells transformed by thymidine kinase gene (TK-gene) of Herpes simplex virus type (HSV 1). The presence of a fragment of human satellite DNA III in the plasmid DNA carrying the TK-gene of HSV 1 reduced notably the rate of the loss of TK+-phenotype, and the treatment of the cells with a tumour promoter--12-o-tetradecanoyl-phorbol-13-acetate--immediately after transformation destabilizes TK+-phenotype of transformant clones. Removal of the eukaryotic carrier DNA for the plasmid DNA without the TK-gene of HSV 1 destabilizes the clone transformant phenotype. Changes in the structure of the plasmid DNA containing no TK-gene of HSV 1 and introduced into cells simultaneously with TK-gene containing plasmids affects the rate of the loss of TK+-phenotype transformed cells.     

A coordinate relationship between theGALK and theTK1 genes of the Chinese hamster

Chinese hamster cells in culture were treated with various concentrations of thymidine, 5-bromodeoxyuridine, trifluorothymidine, and 2-deoxy-D-galactose. Selection was made for deficiencies in the activities of galactokinase and thymidine kinase. Selection in the presence of thymidine, 5-bromodeoxyuridine, and trifluorothymidine was expected to produce clones deficient in thymidine kinase only, whereas those deficient in galactokinase were expected to be selected in the presence of 2-deoxy-D-galactose. However, it was found that clones growing in the presence of these inhibitors were frequently deficient in both enzymes. Or if a clone was deficient in only one, the deficiency frequently was not expected according to the selection procedure. This indicates some sort of coordinate relationship between the two gene loci, GALK and TK1, which specify galactokinase and thymidine kinase, respectively. GALK and TK1 are linked in all primates and rodents in which linkage determinations have been made. It is therefore probable that this linkage has been conserved for a long period of time. It is suggested that the apparent relationship between the two genes shown by the data presented here, as well as by others, supports the conclusion that linkage has been conserved by natural selection and is therefore not fortuitous.     

Comparison of the Frequencies of Spontaneous and Chemically-Induced 5-Bromodeoxyuridine-Resistance Mutations in Wild-Type and Revertant Bhk-21/13 Cells

5-bromodeoxyuridine resistance mutations induced by mutagenesis were studied. The average expression time for induced mutations varied with the concentration of the mutagen ethyl methanesulfonate (EMS). However, a constant number of two generation times was necessary for half maximal expression of induced mutations. Also, induced mutation rates were compared under optimal expression conditions for bromodeoxyuridine, fluorodeoxyuridine and azaguanine resistance markers. Ten independent bromodeoxy-uridine-resistant clones were tested for reversion. Two clones reverted-one spontaneously and the other after mutagenesis. The spontaneous rate of mutation to bromodeoxyuridine resistance, estimated by the fluctuation test, was high in revertant clones (4 x 10(-6) / cell / generation) and low in the wild-type cells (< 3.5 x 10(-8) / cell / generation). A comparison of induced mutation frequencies at variable EMS concentrations showed a single-hit curve for revertant clones and a multihit curve for the wild-type cells. Thymidine kinase activities of resistant clones were usually less than 2% of that of the wild-type clone. Inducibility, thermal stability and intracellular localization of the thymidine kinases of the wild-type cells and of a revertant clone were identical. A low, but significant (P < 0.10), Km discrepancy was observed between enzyme extracts of these lines. The genetic implications of these results are discussed.     

Comparative study of the transformation of various thymidine kinase-deficient human and animal cell lines with the thymidine kinase gene of the Herpes simplex virus

The comparative study of transformation of four thymidine kinase deficient cell lines (mouse mammary carcinoma cell line FS tk-; rat cell line Rat-2tk-; mouse cell line Ltk-, clone D1; human cell line 143tk-) with the thymidine kinase cloned gene of Herpes simplex virus 1 was undertaken. The differences in efficiency and optimal conditions of transformation were shown for these cell lines. The advantages and disadvantages of the cell systems examined for the use in experiments for transformation and cotransformation of cultured cells with isolated genes are discussed.     

Thymidine kinase deficient human cells have increased UV sensitivity in their capacity to support herpes simplex virus but normal UV sensitivity for colony formation

A thymidine kinase deficient (tk-) and two thymidine kinase proficient (tk+) human cell lines were compared for UV sensitivity using colony-forming ability as well as their capacity to support the plaque formation of herpes simplex type 1 (HSV-1). The tk- line (143 cells) was a derivative of one of the tk+ lines (R970-5), whereas the other tk+ line (AC4 cells) was a derivative of the 143 cells obtained by transfection with purified sheared HSV-2 DNA encoding the viral tk gene. 143, R970-5 and AC4 cells showed a similar UV sensitivity for colony-forming ability. In contrast, the capacity to support HSV-1 plaque formation immediately (within 1 h) after UV-irradiation was reduced to a greater extent in the 143 cells compared to the R970-5 and AC4 cells. Capacity curves for plaque formation of the HSV-1: KOS wild-type (tk+) strain were similar to those for the HSV-1: PTK3B mutant (tk-) strain in the 3 cell strains, indicating that the viral tk gene does not influence the ability of HSV-1 to form plaques in UV-irradiated compared to unirradiated human cells. Cellular capacity for HSV-1 plaque formation was found to recover in both tk- and tk+ cells for cultures infected 24 h after UV-irradiation. These results suggest that repair of UV-damaged DNA takes place to a similar extent in both tk- and tk+ human cells, but the kinetics of repair are initially slower in tk- compared to tk+ human cells.     

Selective gene mutation in MEL cells.

MEL cells, undergoing erythroid differentiation and parasynchronized by dimethyl sulfoxide (DMSO) induction, were irradiated with a 3-s pulse of UV light at sublethal dose. A large number of clones deficient in different gene functions are found in the progeny of the treated cells, if the pulse irradiation is performed 18-24 h from the start of DMSO induction. Kinetics of thymidine incorporation into DNA show that the period of sensitivity corresponds to the S phase. The results show that the activities of the tested genes are differently affected depending on the exact time of cell irradiation. Maximum percent inhibition of cells not expressing glucose-6-phosphate dehydrogenase (G-6-PD) (70%) is produced by irradiating at 20 h from the start of DMSO induction; 6-phosphogluconate dehydrogenase (6-PGD) (55%), and hypoxanthine (guanine) phosphoribosyltransferase (HPRT) (33%), at 21 h; hemoglobin (50%), at 22 h. The time difference in the sensitivity to UV light is highly reproducible and has been exploited to isolate, with high efficiency, cellular clones deficient in any one of the tested functions. Determinations of enzymatic activities on cell lysates show that the expression of tested genes is actually altered in cells that, on the basis of cytochemical tests, appear unaffected by UV irradiation. While the production of mutant clones is observed only during the S phase of the cell cycle, immediate statistical damage of the cellular DNA is produced at all times of irradiation. This finding excludes that the two types of phenotypic alterations, blocked or altered gene expression, both propagated in the progeny of the cells as clonal properties, may derive from a preferential alteration of those functions during the S phase.     

Difference in O6-methylguanine methyltransferase activity among transformed NIH3T3 cell clones

We examined the sensitivity to the lethal effects of methylating agents and the O6-methylguanine methyltransferase (MTR) activities of in vitro transformed NIH3T3 cell clones. The sensitivities to the lethal effects of MNNG were not different among all 49 transformed cell clones examined and do not correlate with the MTR activities. All 8 spontaneously transformed cell clones showed the same sensitivities to ACNU as the parental cell line. 2 of 20 transformants induced by UV or MNNG showed higher sensitivities to the ACNU although the MTR activity was normal. One cell clone transformed by UV was sensitive to ACNU and showed about half MTR activity. 5 of 19 cell clones transformed by oncogenes (Ha-ras or SV40 ori-) were sensitive to the lethal effects of ACNU and showed the low MTR activities, but were not as much sensitive as a Ha-MuSV transformed cell clone, Ha821.     

Role of the viral and cellular encoded thymidine kinase in the repair of UV-irradiated herpes simplex virus

A strain of herpes simplex type 1 (HSV-1:KOS) encoding a functional thymidine kinase (tk+) gene and a thymidine kinase deficient (tk-) mutant strain (HSV-1:PTK3B) were used as probes to examine the repair of UV-damaged viral DNA in one tk- (143) and two tk+ (R970-5 and AC4) human cell lines. UV survival for each HSV-1 strain was similar for infection of both tk- and tk+ cells suggesting that the repair of viral DNA was not dependent on the expression of a functional cellular tk gene. In contrast, UV survival of HSV-1:PTK3B was substantially reduced compared to HSV-1:KOS when infecting all 3 human cell lines, as well as Vero monkey kidney cells and LPM1A mouse cells. These results suggest that the repair of UV-irradiated HSV-1 in lytically infected mammalian cells depends, in part at least, on the expression of the viral encoded tk.     

Isolation of UV-sensitive variants of human FL cells by a viral suicide method.

A new method (viral suicide method) for the isolation of UV-sensitive mutants is described. Colonies of mutagenized human FL cells were infected with UV-irradiated Herpes simplex viruses and surviving ones which seemed to be deficient in host cell reactivation (HCR) were examined for their UV sensitivity. Nineteen of 238 clones examined were sensitive to UV irradiation at the time of the isolation. After recloning, four of these clones have been studied and two (UVS-1 and UVS-2) of them are stable in their UV sensitivity for 4 months in culture. UV sensitivity of UVS-1, UVS-2, and the parental FL cells are as follows: the extrapolation numbers (n) are 2.2, 2.1, and 1.8 and mean lethal doses (D0) are 2.9, 3.7, and 7.8 J/m2 for UVS-1, UVS-2, and the parental FL cells, respectively- They are no more sensitive than FL cells to X-irradiation. The ability of HCR in UVS-2 cells is apparently lower than that in FL cells, whereas UVS-1 cells are the same as FL cells in the ability.     

Genetic transformation of somatic cells. I. Clone of Chinese hamster cells defective in thymidine kinase and characterized by high transformation efficiency

A characteristics is given of clone A238 of the Chinese hamster cells deficient in thymidine kinase (TK). The isolation procedure is described. Upon transformation with the aid of DNA of plasmids, containing thymidine kinase gene (tk-gene) of Herpes simplex virus type 1 (HSV1) clone A238 cells show frequency (7.10(-5) and efficiency (130 TK+ colonies per 1 microgram of plasmid DNA) compatible with those of mouse line LMtk- cells. Modified transformation and selection conditions of clone A238 cells expressing TK-gene of HSV1 are demonstrated. A simple method is described for discriminating somatic cells, expressing either their proper or a virus TK-gene according to the cloning efficiency of cells on the HAT medium containing thymidine in concentration 100 micrograms/ml. It is shown that at the fixed total DNA concentrations a complete replacement of the eukaryotic carrier DNA for the plasmid DNA, containing no tg gene of HSV1, decreases but only insignificantly the frequency and efficiency of transformation.     

Mouse thymidine kinase: the promoter sequence and the gene and pseudogene structures in normal cells and in thymidine kinase deficient mutants.

The mouse genome carries one gene and two pseudogenes for cytoplasmic thymidine kinase. The overall structure of these genes was determined with the help of cosmids and lambda phage clones and the upstream sequence containing the promoter was determined. The data allow an allocation of bands seen in the complex patterns of genomic Southern blots obtained from the DNA of wild type cells and of thymidine kinase deficient mutants to the gene as well as to the two pseudogenes. The much used LTK cell line was found to lack the entire gene but to retain the pseudogenes. Two other TK cell lines had DNA patterns indistinguishable from the wild type. Whereas the LTK line did not produce any TKmRNA, the two other mutants had normal amounts of TKmRNA but no cytoplasmic TK activity.     

Characteristics of spontaneous and induced thymidine and 5-iodo-2-deoxyuridine resistant clones of mouse lymphoma cells

Clones resistant to 5-iodo-2-deoxyuridine (IUdR) were isolated from P388 cells and cultured in the absence of selective medium. Thymidine kinase assays were performed on 8 clones which had arisen spontaneously and 19 isolated after exposure to X-rays or alkylating agents. All the clones tested showed significantly reduced thymidine kinase activity relative to wild-type cultures, but none showed zero levels. 14 of these clones were tested for thymidine (TdR) uptake and all showed a marked reduction in the rate of [³H]TdR incorporation into acid soluble fractions and into DNA. 7 IUdR-resistant (IUdR^r) clones were tested for revertibility as measured by growth of colonies in HAT medium. 5 of the 7 were found to revert at measurable rates either spontaneously or after a low dose of mutagen.Thymidine kinase activity was also measured in 8 thymidine resistant P388 clones (TdR^r). Initial rates of thymidine phosphorylation were not significantly altered in 5 of the 8 clones tested but significantly lower amounts of phosphorylated products were observed in 6 of the 8 clones. [³H]TdR uptake was reduced in 9 of 12 clones tested, and 2 of them showed no corresponding reduction in the thymidine kinase activity, suggesting the occurence of mutants with altered permeability for thymidine.IUdR resistant L5178Y clones could not be isolated. Thymidine resistant L5178Y clones were similar to TdR^r P388 clones, i.e. they showed changes in initial rates of thymidine kinase activity and reduced accumulation of phosphorylated products. Only one clone could be shown to be a membrane mutant. These results are discussed in relation to the genetic nature of the thymidine kinase locus in the two cell lines.     

Genetic transformation of somatic cells. VII. The loss of the transformant phenotype is accompanied by both stable and unstable changes in DNA

From 6 clones of Chinese hamster cells varying in the rate of the loss of transformant phenotype and containing a thymidine kinase gene (tk-gene) of Herpes simplex virus type 1 (HSV1), 25 subclones negative in thymidine kinase (TK-) were isolated on a medium with 50 micrograms/ml 5-bromodeoxyuridine (BrdU). A study was made of the frequency of spontaneous reversions to the TK+ phenotype in cell populations of BrdU-resistant subclones, and of the transforming activity (upon transformation of TK- cells of A238 clone to the TK+ phenotype) of DNA preparations from a row BrdU-resistant subclones. In 7 of 11 BrdU-resistant subclones the TK- phenotype is associated with changes reducing significantly the transforming activity of DNA. Some of these alterations are stable and undergo no spontaneous reversion, while the other ones are unstable, being reversed or suppressed at a high frequency. BrdU-resistant subclones produced from clones more stable in transformant phenotype are on the whole more stable in the TK- phenotype than BrdU-resistant subclones from the clones with the high rate of the loss of the TK+ phenotype.     

Mutagenesis, by methylating and ethylating agents, in mutH, mutL, mutS, and uvrD mutants of Salmonella typhimurium LT2.

Salmonella typhimurium LT2 mutH, mutL, mutS, and uvrD mutants were especially sensitive to mutagenesis by both the recA+-dependent mutagen methyl methane sulfonate and the recA+-independent mutagen ethyl methane sulfonate, but not to mutagenesis by agents such as 4-nitroquinoline-1-oxide and UV irradiation. Similarly, these mutator strains were very sensitive to mutagenesis by the methylating agents N-methyl-N'-nitro-N-nitrosoguanidine and N-methyl-N-nitrosourea. The increased susceptibility to mutagenesis by small alkylating agents due to mutH, mutL, mutS, and uvrD mutations was not accompanied by an increased sensitivity to killing by these agents. Various models are discussed in an effort to explain why strains thought to be deficient in methyl-instructed mismatch repair are sensitive to mutagenesis by methylating and ethylating agents.     

Induction of gene mutations and the lethal action of ultraviolet rays in synchronized Chinese hamster cells

Lethal and mutagenic effects of UV light were studied in two synchronized UV-sensitive Chinese hamster cell clones differing in the degree of sensitivity (CHS1, CHS2). It is shown that the phase of mitosis is most resistant to the lethal effect of UV. The sensitivity of both cell clones increases in the pre-synthetic phase and reaches its maximum during the phase of DNA synthesis. Positive correlation of cell sensitivity to mutagenic and lethal action of UV was observed when studying induced mutability in both cell clones during the phase of DNA synthesis. However, the study of the mutagenic effect of UV on different phases of the synthesis. However, the study of the mutagenic effect of UV on different phases of the cell cycle (M, G1, S) in the less UV-sensitive cell clone has revealed that the maximal mutation yield takes place when cells are irradiated at G1 (CHS1). The discrepancy observed may be due to different probability of the phenotypic detection of pre-mutational lesions, arising at different phases of the cell cycle. It is shown that only one cell generation is necessary for the expression of pre-mutational changes. These data allow to conclude that the increased mutation rate observed at G1 (as compared with S) reveals rather a probability of the expression but not of the occurrence of pre-mutational lesions. It is suggested that the fixation of mutations in the cells studied proceeds during the post-replication repair synthesis.     

Selective inhibition of the proliferation of herpes simplex virus type 1 thymidine kinase gene-transformed murine mammary FM3A carcinoma cells by (E)-5-(2-bromovinyl)-2'-deoxyuridine and related compounds

Mouse mammary carcinoma FM3A cells deficient in thymidine kinase were transformed by a cloned gene for herpes simplex virus type 1 thymidine kinase. Among several anti-herpetic nucleoside analogues, (E)-5-(2-bromovinyl)-2'-deoxyuridine, (E)-5-(2-iodovinyl)-2'-deoxyuridine and (E)-5-(2-bromovinyl)-2'-deoxycytidine inhibited the growth of the transformed cells at concentrations 5000- to 20000-fold lower than those required to inhibit the growth of the corresponding wild-type cells. The selective inhibitory action of these compounds was due to a specific phosphorylation by the viral thymidine kinase. From the transformed cells, thymidine-auxotrophic mutants that are deficient in thymidylate synthase were isolated. These mutant cell lines should prove useful in elucidating the mechanism of action of the antiherpetic nucleoside analogues.