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Objective: To identify adults and children as under- (UR), acceptable (AR), or over-reporters (OR) of energy intake (EI) using energy expenditure measured by doubly labelled water (DLW) (EE(DLW)), and to use this as a reference to determine the sensitivity and specificity of (i) EE measured by heart rate (EE(HR)), and (ii) the Goldberg cut-off technique for classifying subjects into the same categories., Design: Retrospective analysis of a dataset comprising concurrent measurements of EE(DLW), EE(HR), basal metabolic rate (BMR), and EI by weighed record (EI(WR)) on 14 adults and 36 children. EI by diet history (EI(DH)) was also measured in the children only. EI(WR):EE(DLW) provided the reference definition of subjects as UR, AR or OR. Three strategies for classifying mis-reporters based on EE(HR) and Goldberg cut-offs were then explored. Sensitivity and specificity were calculated respectively as the proportion of UR and non-UR correctly identified., Results: Approximately 80% of all subjects were AR. For EI(WR) and EI(DH) respectively, the sensitivity of EE(HR) was 0.50 and 1.00, and specificity was 0.98 and 1.00. Although designating subjects as having low, medium or high activity levels (EE(HR):BMR(meas)) and calculating cut-offs based on appropriate WHO physical activity level PALs did not change sensitivity, specificity dropped to 0.98 (EI(WR)) and 0.97 (EI(DH)). Cut-offs based on a PAL of 1.55 reduced sensitivity to 0.33 (EI(WR)) and 0.00 (EI(DH)), but specificity remained unchanged. The sensitivity of all cut-offs based on physical activity level (PALs) for EI(WR) was 0.50 (adults) and 0.25 (children)., Conclusions: If the precision of EE(HR) was improved, it may be useful for identifying mis-reporters of EI.

Nucleotide pool imbalances have been reported to affect the fidelity of DNA replication and repair in prokaryotic and eukaryotic cells. We have reported previously that the mutagen-hypersensitive thymidine kinase (TK)-deficient Friend erythroleukemia (FEL) cells (subclones 707BUF and 707BUE), have a more than sixfold increase in the dCTP:dTTP pool ratio when compared to that of wild-type, TK-positive (TK(+)) clone 707 cells. In this study we present the results of an investigation of the effect of the dCTP:dTTP pool imbalance on the accuracy of DNA replication within 707BUF cells. We examined the spontaneous mutation spectra occurring at the adenine phosphoribosyltransferase (aprt) locus within clone 707 (TK(+)) and 707BUF (TK(-)) FEL cells. Mutations recovered at the aprt locus in FEL cells comprised: base substitutions (43:73), frameshifts (14:13.5), and deletions (43:13.5) [clone 707 (TK(+)):707BUF (TK(-)), respectively, expressed as percentages]. A comparison of the mutation spectra obtained for the two cell lines did not reveal any significant increase in misincorporation of dCTP, the nucleotide in excess, in 707BUF (TK(-)) cells, during DNA replication synthesis. These data suggest that the dCTP:dTTP pool imbalance does not alter the fidelity of DNA replication synthesis in 707BUF (TK(-)) FEL cells. Rather, the predominance of GC --> AT transitions (53%) in the 707BUF (TK(-)) spectrum may reflect a reduced efficiency of repair by uracil DNA glycosylase of uracil residues within these cells., (Copyright 2000 Wiley-Liss, Inc.)

Deficiencies of antioxidant nutrients have been implicated in the etiology of lung and other cancers. However, most intervention trials with antioxidant nutrients have not shown beneficial effects, and some have indicated that beta-carotene may be deleterious. This randomized, double-blind, placebo-controlled study evaluated the effects of five short-term (4-wk) antioxidant nutrient supplement regimens [ascorbic acid (350 mg), RRR-alpha-tocopherol (250 mg), beta-carotene (60 mg), selenium (80 micrograms as sodium selenite), ascorbic acid (350 mg) + RRR-alpha-tocopherol (250 mg)] on plasma antioxidants and mononuclear leukocyte DNA damage in male smokers (n = 9) and nonsmokers (n = 12). Plasma concentrations of ascorbic acid and tocopherol were significantly increased by supplementation, but there was no significant change in plasma beta-carotene or blood glutathione peroxidase activity after supplementation with beta-carotene or selenium. DNA damage in mononuclear leukocytes, as assessed by comet assay, was not affected by any supplementation regimen. DNA damage, as assessed by 8-hydroxydeoxyguanosine in mononuclear leukocytes, was not influenced by ascorbic acid, alpha-tocopherol, or selenium supplementation in smokers or nonsmokers, but beta-carotene supplementation resulted in significant differences between smokers and nonsmokers in the level of oxidative DNA damage, with decreases in smokers and increases in smokers. This is a further indication of the differential effects of supplemental beta-carotene in smokers and nonsmokers.

In this study, the effect of thymidine kinase deficiency on the responses of the human lymphoblastoid cell line Raji to methyl methanesulphonate and mitomycin C was investigated. Mutagen sensitivity was measured in terms of cell survival and mutation to hypoxanthine-guanine phosphoribosyltransferase deficiency. Thymidine kinase-deficient Raji cells showed decreased survival and increased mutant frequency relative to wild-type cells following treatments with each of the mutagens used. It is suggested that this may be due to an imbalance in the supply of deoxyribonucleoside triphosphates to the excision repair process. The role of O6-methylguanine-DNA methyltransferase in the repair of DNA damage caused by these mutagens is also discussed.

Deficiency of the enzyme adenine phosphoribosyltransferase (APRT) has been associated with hypersensitivity to the mutagenic effects of ethyl methanesulphonate (EMS) and 254 nm ultraviolet (UV) radiation in clone 707 of Friend mouse erythroleukaemia (FEL) cells. The molecular nature of spontaneous EMS- and UV-induced mutations in the coding region of hypoxanthine-guanine phosphoribosyltransferase (HPRT) was determined for wild-type FEL cells and two APRT-deficient mutant sub-clones which have significantly reduced ATP pool levels, and are mutagen-hypersensitive. Mis-sense base substitutions were the predominant type of spontaneous mutation. However, exon deletions, possibly involving aberrant splicing of HPRT mRNA, and a non-sense mutation were also observed. EMS-induced mutations in wild-type and APRT-deficient mutant sub-clones were GC-->AT transitions, which is consistent with O6-ethylguanine being the primary pre-mutagenic lesion. All UV-induced mutations in both cell types were targeted to dipyrimidine sites where the two most common classes of photoproducts (cyclobutane pyrimidine dimers and [6-4] photoproducts) are formed. The similarity in the mutations observed in both cell types indicates that the mutagen hypersensitivity of APRT-deficient cells may be the result of decreased efficiency in the excision repair processes due to reduced levels of ATP.

The alkaline comet assay or single cell microgel electrophoresis assay is a sensitive method of detecting DNA strand breaks and alkali labile sites in individual cells. The results of this assay can be analysed by different methods. In this study we compared analyses of the same slides by a manual method and by image analysis, post-treatment of clone 707 Friend erythroleukaemia cells with H2O2. The parameters which were found to be particularly useful were comet area and comet length (measured manually) and percentage tail DNA, tail moment, tail length and tail length/head radius (L/H), measured using image analysis. The manual method for comet analysis presented in this paper would appear to provide good and reliable comet data. However, the image analysis comet system described offers an alternative analysis method which avoids the need for photomicrographs and tedious manual analysis. The image analysis parameters: % tail DNA, tail moment, tail length and L/H give good consistent results and for large-scale analysis it will, therefore, conceivably be the method of choice.

HL-60 and U-937 cells were used as models to assess the involvement of the enzymes of thymidine metabolism in differentiation. Both cell types showed decreased thymidine kinase and thymidylate synthase but increased thymidine phosphorylase activities in response to the induction of differentiation by dimethylsulfoxide and 12-O-tetradecanoylphorbol 13-acetate. This was accompanied by a greater than three-fold increase in the stimulation of superoxide production in both cell lines. Thymidylate synthase and thymidine kinase activities were noted as potential markers of leukaemic cell proliferation while thymidine phosphorylase and superoxide production correlated well with differentiated phenotypes. Prolonged treatment of U-937 by 12-O-tetradecanoylphorbol 13-acetate resulted in a marked de-differentiation, indicating overstimulation of one or more of the isoforms of protein kinase C.

In this paper the DNA damage and repair induced by the radiomimetic agent bleomycin are compared in murine Friend erythroleukaemia wild-type 707 cells and a thymidine kinase-deficient sub-clone BUF. Comparisons are made using results obtained from the alkaline comet assay and unscheduled DNA synthesis experiments. Further analysis to determine the fidelity of bleomycin-induced repair as indicated by mutagenesis to hypoxanthine-phosphoribosyltransferase deficiency was also conducted. Similar sensitivities to bleomycin treatments were observed in the two cell types with the comet assay, while similar levels of dose-dependent excision repair following bleomycin treatments were also detected in unscheduled DNA synthesis experiments. Comet assay and unscheduled DNA synthesis experimental results are in agreement. Survival and induced hypoxanthine-phosphoribosyltransferase mutant frequencies were observed to be unaffected by a thymidine kinase-deficiency in Friend erythroleukaemia cells. The results of this investigation suggest no overall difference in the repair capacities or the repair fidelity of Friend 707 relative to BUF cells following bleomycin treatments.

Thymidine kinase (TK), an enzyme known to be associated with DNA synthesis, has been measured in the serum of patients with asymptomatic colorectal adenomas (n = 40), asymptomatic colorectal carcinoma (n = 21) and patients known to have hepatic metastases form colorectal tumours (n = 33); enzyme levels have been compared with an age-matched group of asymptomatic people with no evidence of colorectal neoplasia at screening colonoscopy (n = 26). TK activity in patients with metastatic disease (median 4.23; range 2.03-14.12 pmol/ml/h) and in patients with adenomas (median 2.33; range 1.59-8.73 pmol/ml/h) was significantly higher than in the normal controls (median 2.04; range 1.29-5.40 pmol/ml/h). However TK activity in patients with asymptomatic cancer (median 1.85; range 1.00-4.50 pmol/ml/h) was not significantly different from the control group.

The purpose of this study was to investigate the cytotoxic and genotoxic potential of low intensity laser irradiation (660 nm, 12 mW, 5 kHz) on mammalian cells. Thymidine kinase (TK)-positive and TK-deficient Friend erythroleukaemia (FEL) cells, clone 707 and subclone 707BUF respectively, were used in this investigation. Following irradiation of exponentially growing cells in suspension at doses of 2 and 20 J/cm2 a number of sensitive bioassays were used to facilitate the detection of laser-induced mutations, DNA damage and cell killing. Mutations were assessed by the examination of chromosome spreads, the determination of micronucleus frequency and by the determination of the mutant frequency at the hypoxanthine-guanine phosphoribosyltransferase (hgprt) locus. DNA damage was quantified using a sensitive ELISA. The cytotoxic effect of laser irradiation was assessed using a cloning assay. The results of this investigation did not show any significant increase in mutation frequency, DNA damage or cell survival in the laser-irradiated cells, compared to sham-irradiated controls. The lack of any demonstrable cytotoxic and genotoxic effects of low intensity laser irradiation on mammalian cells in culture would support it as being a safe modality for clinical use.

In this study the effect of thymidine kinase (TK) deficiency on mutagen sensitivity was examined in the human lymphoblastoid cell line Raji. Wild-type and TK-deficient Raji cells were treated with a range of concentrations of ethyl methanesulphonate (EMS) and a range of doses of ultraviolet (UV) light, then examined for mutagen sensitivity as measured by cell survival and mutation to HGPRT deficiency. Dose-dependent responses were observed and TK-deficient cells exhibited decreased survivals and increased mutant frequencies relative to wild-type cells. TK-deficient Raji cells are also deficient in O6-methylguanine-DNA-methyltransferase. This may partially account for their sensitivity to EMS but does not account for the results obtained with UV. It is therefore likely that an additional factor, such as alterations in supply of deoxyribonucleoside triphosphates, may affect the mutagen sensitivity of Raji cells.

An accumulation of mutations on their own or together with other age-related changes may contribute to aging and the development of age-related pathologies. The aim of this investigation was to assess the extent of DNA mutations as a function of age in humans. The mutant frequency (MF) at the hypoxanthine-guanine phosphoribosyl-transferase (hgprt) locus was assessed in lymphocytes isolated from male volunteers in each of three age groups (35-39, 50-54 and 65-69 years). Results show that the mean MF in the 65-69 years group was approximately twice that in the 35-39 and 50-54 years groups (4.1/10(6) cells, 1.9/10(6) cells and 1.79/10(6) cells, respectively) increasing by about 1.33% per year, after 54 years. In addition, there was an increased frequency of chromosomal aberrations in the 65-69 years group compared to the other two age groups. The results of this investigation show an increase in DNA mutations in cultured human lymphocytes with age. Factors which may influence the extent of DNA damage in human lymphocytes are discussed.

Thymidine kinases (TK) convert thymidine, or deoxythymidine (dT) to the respective monophosphate. TK occurs in many different procaryotic and eucaryotic species and different TK isoenzymes are found within the same eucaryotic cell. One isoenzyme (foetal, cytoplasmic, TK1) is associated with cell division while the other (adult, mitochondrial, TK2) is cell cycle independent. The relative isoenzyme activities in a tissue thus reflect the fraction of proliferating cells. The gene encoding TK1 has been cloned for many species and regulation of its expression is known to be complex. Increases in TK activity appear to correlate with the presence of human neoplasia and disease progression and regression have been reported to correlate with TK levels in many cancer types. TK estimations in human lymphoproliferative diseases have implicated this enzyme as an early marker of maldifferentiation. TK levels may also be increased in non-dividing mammalian cells infected with RNA or DNA viruses. Some virus encoded TK has been shown to differ biochemically, immunologically and in substrate specificity from the corresponding TK isoenzymes in target host cells thus facilitating the development of specific antiviral therapeutics. Further, TK1 in leukemic cells may differ biochemically from normal cellular TK1 such that tumor-specific TK may provide a target for tumor detection and therapy. TK quantitation has conventionally been performed in assays of enzyme activity using radiolabeled (3H or 125I) nucleoside substrates. The development of TK1-specific, non-radioisotope based immunoassays and the measurement of TK mRNA in tumour tissue using TK (DNA or RNA) probes may prove sufficiently valuable to be incorporated into the routine clinical management of human cancer.

The majority of primary bladder neoplasms are known to arise within the mucosa around the ureteric orifices and bladder base. This may be due to the mucosa in this area being more susceptible to carcinogens than other areas of the bladder. Deficiency in the nucleotide salvage pathway enzyme thymidine kinase (TK), and especially its TK1 isozyme, has been shown to predispose cell lines to increased mutagenesis. Total TK and TK1 activities were measured in mucosal samples taken adjacent to the ureteric orifices and dome in 32 normal bladders and both total TK and TK1 were shown to be significantly decreased in the mucosa adjacent to the ureteric orifices. This may explain why primary bladder neoplasms occur more commonly in this site.

Thymidine kinase (TK) exists in two forms, TK1 and TK2. TK levels and oestrogen receptor status (OR) were measured in tumours from 86 patients with operable breast cancer and the patients were monitored for recurrence over 24 months. During the monitored period, 13 patients showed recurrence. These patients also exhibited higher total TK levels per mg tumour (P < 0.01) and higher TK1 levels (P < 0.001) than those who did not show recurrence. TK1 levels relative to TK2 were significantly higher (P < 0.05) in OR-negative tumours (n = 29) than in OR-positive tumours (n = 57). OR-negative (n = 9) and OR-positive (n = 4) patients who recurred had significantly higher TK1 levels relative to TK2 than did those who not recur (n = 20 and n = 53, respectively). These preliminary results indicate that breast tumour TK levels may have value in determining prognosis.

HLA-DPB1 allele frequencies in 150 unrelated normal individuals from Northern Ireland were determined using oligonucleotide typing methods. HLA-DPB1*0401 was the most common allele in the population possessed by 75.3% of subjects, followed by DPB1*0201 (20.7%). In addition to these alleles, only HLA-DPB1*0402, -DPB1*0301, and -DPB1*0501 were present in subjects at frequencies greater than 10%. The results in this study are in broad agreement with other Caucasoid studies, but there is regional and ethnic variation in HLA-DP allele frequencies. Three DPB1 alleles were found to be in linkage disequilibrium with HLA-DR antigens determined by RFLP, namely, DPB1*0101 with DRw17 (Dw24 associated) RFLP, DPB1*0501 with DRw13-Dw19 RFLP, and DPB1*1901 with DRw13-Dw18 (Dw25 associated) RFLP. One individual revealed a novel DPB1 pattern of probe reactivity, which following DNA sequencing was found to be HLA-DPB1*2001. To assess the system used and to compare consistency of results between laboratories, 62 cell lines were oligotyped for HLA-DP. The results revealed the system described here to be extremely accurate and showed excellent agreement of HLA-DP typing results for cell lines between laboratories.

A wide range of DNA damage is known to be caused by reactive oxygen species (ROS). Defence against the effects of such damage include damage prevention (e.g. antioxidant activity) and the removal of damaged moieties from DNA (DNA repair). Radiation (X-ray) sensitive murine lymphoma (LY) cells were seen to be more susceptible to ROS-induced damage than were radiation resistant cells. This difference was unlikely to be due to the marginally decreased DNA excision repair capacity of the sensitive cells. Radiation sensitive cells did, however, have lower endogenous antioxidant enzyme levels. Thus, the importance of assessing all levels of a cell's response to ROS, in determining the major factors leading to increased mutagen sensitivity, is emphasised.

Total daily energy expenditure (TEE) by the doubly-labelled (2H218O) water method and basal metabolic rate (BMR) by indirect calorimetry were measured in thirty-two healty free-living adults in Northern Ireland. Habitual physical activity patterns in occupational and discretionary activities were assessed by interview questionnaire. Expressed as a multiple of BMR the TEE values for the sixteen males (1.88 (SD 0.28), range 1.44-2.57) and sixteen females (1.77 (SD 0.16), range 1.50-2.06) were compatible with current Department of Health and Social Security (DHSS; 1979) and Food and Agriculture Organization/World Health Organization/United Nations University (FAO/WHO/UNU; 1985) estimates of energy requirements. The results suggest that discretionary physical activity is now emerging as an equally important determinant of energy expenditure in the UK as the occupational classifications currently used as the basis of DHSS (1979) and FAO/WHO/UNU (1985) recommendations for energy requirements. Therefore, realistically achievable inputs of recreational exercise can have a significant impact in counteracting low levels of energy expenditure which are associated with modern lifestyles and are implicated as a risk factor for coronary heart disease and obesity.

Wild-type Friend mouse erythroleukaemia cells (clone 707) were compared with adenine phosphoribosyltransferase (APRT)-deficient mutant subclones (707DAP8 and 707DAP10) for sensitivity to cell killing and mutagenesis by ethyl methanesulphonate (EMS) and methyl methanesulphonate (MMS). Cells were exposed to 0-300 micrograms/ml EMS and to 0-20 micrograms/ml MMS for a period of 16 h. A slight difference was found between wild-type cells and the two APRT-deficient subclones in terms of sensitivity to cell killing by both mutagens. The APRT-deficient subclones were, however, significantly more sensitive than wild-type cells to mutagenesis to 5-bromo-2-deoxyuridine resistance and 6-thioguanine resistance by EMS and MMS. The APRT-deficient subclones were found to have significantly decreased levels of dATP and dTTP nucleotides and decreased levels of all four ribonucleoside triphosphates (ATP, GTP, CTP and UTP) relative to wild-type cells. Wild-type Friend cells were found to have insignificant levels O6-methylguanine-DNA methyl transferase and it is suggested that the increased mutagen sensitivity of APRT-deficient cells may be due to imbalance of deoxyribonucleoside triphosphate pools during DNA excision-repair processes, or more probably due to deficiency of ATP for ATP-dependent DNA excision-repair enzymes.

Objective: To provide an independent evaluation of seven day weighed dietary records, which are currently accepted as the most accurate technique for assessing habitual dietary intake in studies investigating the links between diet and health., Design: Subjects who had previously participated in the Northern Ireland diet and health study were reselected by stratified random sampling to represent the range of energy intakes in the study as assessed by the seven day weighed dietary record., Setting: Northern Ireland., Subjects: 31 Free living adults (16 men and 15 women)., Main Outcome Measures: Energy intake as measured by the seven day weighed dietary record and total energy expenditure estimated concurrently by the doubly labelled water technique., Results: Average recorded energy intakes were significantly lower than measured expenditure in the group overall (9.66 MJ/day v 12.15 MJ/day, 95% confidence interval 1.45 to 3.53 MJ/day). Among those in the upper third of energy intakes the mean (SE) ratio of intake to expenditure was close to 1.0, indicating accurate records (men 1.01 (0.11), women 0.96 (0.08]. In the middle and lower thirds the ratios for men were only 0.74 (0.05) and 0.70 (0.07) respectively and for women 0.89 (0.07) and 0.61 (0.07)., Conclusions: These results show a serious bias in reporting habitual energy intake. If substantiated they may have wide implications for the interpretation of many nutritional studies.

Wild-type (clone 707) Friend murine leukaemia cells were compared with two ara-C-resistant subclones in terms of sensitivity to cell killing and mutagenesis to 6-thioguanine resistance following treatment with ethyl methane sulphonate, methyl methane sulphonate and UV irradiation. The ara-C-resistant subclones, 707DKE and 707DK48, had respective deoxycytidine kinase activities of 6.7 and 5.4% the values found in wild-type cells. No clear pattern of altered sensitivity to cell killing or mutagenesis emerged between the wild-type cells and the ara-C-resistant subclones. These results do not provide evidence for a role of deoxycytidine kinase in determining sensitivity to mutagenic agents in the Friend cell line.

Clone 707 of the Friend cell was compared with an APRT-deficient subclone for sensitivity to cell killing and the induction of cytogenetic aberrations by mitomycin C (MMC). Two 16-h doses of MMC were used, 0.1 and 0.15 microgram/ml and cells were scored for aberrations at 16, 33 and 44 h post-treatment. The APRT-deficient subclone showed increased cell killing, a higher frequency of aberrations and a higher frequency of cells with severe cytogenetic damage. It is proposed that APRT may play a role in balancing deoxyribonucleoside triphosphate pools for DNA-repair processes.

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 gamma 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 resistance 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.

The thymidine kinase-deficient subclone, 707BUF, of the Friend murine leukaemia cell line exhibits increased sensitivity to the induction of cytogenetic aberrations by mitomycin C (MMC) relative to wild-type clone 707. It has been suggested that thymidine kinase-deficient cells may be highly mutagen-sensitive through an imbalance of nucleotide pools rendering excision repair error-prone. In this study clone 707 Friend leukaemia cells were compared with subclone 707BUF for sensitivity to the potentiating effect of caffeine on MMC-induced cytogenetic aberrations. The results indicate that although potentiation of mitomycin C-induced cytogenetic damage occurs in both clone 707 and in subclone 707BUF following caffeine treatment, the mutagen-sensitive thymidine kinase-deficient subclone 707BUF had enhanced potentiation by caffeine of MMC-induced cytogenetic damage relative to wild-type clone 707. It is suggested that caffeine may enhance mutagen sensitivity by inhibiting post-replication repair processes and may perhaps also indirectly reduce the effectiveness of the excision repair system by inhibiting the mutagen-induced G2-delay. Clone 707 wild-type cells in the presence of caffeine could then continue to repair DNA damage through an intact though less effective excision repair system, whilst the thymidine kinase-deficient subclone 707BUF would, in the presence of caffeine, be rendered highly mutagen sensitive, being only able to repair DNA damage through an error-prone excision repair process.

Wild type Friend erythroleukaemia cells (clone 707) and thymidine kinase (EC. 2.7.1.2.1) deficient derivatives were examined for sensitivity to killing by ultra-violet (UV) irradiation. Deficiency of thymidine kinase leads to increased cell killing as evidenced in all of twelve thymidine kinase deficient clones examined. A revertant thymidine kinase positive clone was found to have a level of thymidine kinase activity and UV sensitivity intermediate between wild type cells and its thymidine kinase deficient parent clone. The increased cell killing in thymidine kinase deficient clones is also reflected in increased mutagenesis by UV to 6-thioguanine resistance. It is suggested that the increased mutagenesis and sensitivity is due to defective DNA repair as a result of an imbalance in deoxyribonucleoside triphosphate pools in thymidine kinase deficient cells.

A detailed cytogenetic investigation was carried out on P388 mouse lymphoma cells. The cells have a mean chromosome number of 36.86 with a mode and median of 37 chromosomes. G-banding analysis of 12 spreads revealed a total of 15 marker chromosomes with chromosome 11, the determinant of thymidine kinase, being present only in single copy per cell. It is therefore concluded that the P388 cell line is hemizygous at the thymidine kinase locus. Thymidine kinase activities were assayed in P388 cells and two other malignant cell lines, clone 707 Friend mouse leukaemia cells and L5178Y mouse lymphoma cells. No clear relationship was observed between enzyme activity and gene dosage.

Clone 707 of the Friend leukaemia cell line was compared with the hypermutable thymidine kinase deficient subclone 707 BUF for sensitivity to the induction of cytogenetic aberrations by mitomycin C (MMC). Two 16-h doses of MMC were utilized, namely 0.1 and 0.15 microgram ml-1. Following removal of MMC from the cultures, metaphase spreads were prepared after 15, 29 and 43 h growth in non-selective medium. Thirteen types of aberrations were scored. The thymidine kinase deficient subclone showed considerably increased sensitivity to the induction of aberrations, with the aberrations also persisting longer. In light of these and earlier reported results, the significance of thymidine kinase for accurate DNA repair is discussed.

The cytogenetic aberration-inducing effect of excess thymidine and U.V. light in Friend erythroleukaemia (clone 707) cells was investigated. Three doses of U.V. were utilised, namely 2.4, 4.8 and 7.2 J/m2. Thymidine, at 1 X 10(-5) M, was present for 48 h prior to U.V. treatment and for 15 h following it. Although no significant increase in metaphase aberrations was observed following thymidine treatment alone, relative to the spontaneous frequency, clone 707 exhibited increased sensitivity to U.V.-induced cytogenetic damage, when grown in the presence of 1 X 10(-5) M thymidine. The observed synergism between U.V. and thymidine treatments may be due to thymidine-induced nucleotide pool imbalance with consequent inaccuracies in DNA repair.

The abilities of wild-type and thymidine kinase-deficient Friend mouse erythroleukemia cells to perform unscheduled DNA synthesis (UDS), through the incorporation of [3H]deoxycytidine, were measured following damage with methyl methane sulfonate (MMS), ethyl methane sulfonate (EMS), and ultraviolet (UV) irradiation. For each mutagenic treatment, a positive and quantitatively similar response was observed for both wild-type and thymidine kinase-deficient cells. The extent of the response varied greatly, however, depending upon the mutagen used. The results contrast with the unscheduled incorporation of [3H]thymidine in wild-type cells following mutagen treatment, where less variation between the positive UDS responses elicited by MMS, EMS, and UV treatments was observed. Nevertheless, the results clearly indicate that thymidine kinase deficiency does not prevent excision repair (UDS) from occurring.