Biological effects of incorporation of O-methyldeoxyguanosine into Chinese hamster V79 cells

Analysis of the biological effects of specific DNA alkylations by simple alkylating agents is complicated by the variety of sites involved. It is, therefore, of value to be able to incorporate into cellular DNA nucleosides alkylated in a single position, e.g., O⁶-methyldeoxyguanosine. Such cellular incorporation is particularly difficult to achieve because this nucleoside is rapidly demethylated by adenosine deaminase. We have attempted to achieve such incorporation into the DNA of V79 cells by using coformycin, an inhibitor of adenosine deaminase, and by forcing the cells to depend on exogenous purines by the use of medium containing aminopterin. The DNA of V79 cells exposed to O⁶-methyl-[8-³H]deoxyguanosine (2.4 μM, sp. act. 14 500 Ci/mole) showed an incorporation level of 4 × 10⁻⁸ nucleotides. When 1000-fold higher concentrations were employed (3–15 mM, sp. act. 1.6 Ci/mole), significant cytotoxicity and inhibition of DNA synthesis was observed. However, because it was not economically feasible to administer high specific activity O⁶-methyldeoxyguanosine to the cells at these concentrations, we could not determine the amount of labeled nucleoside incorporated into DNA. Examination of the frequency of 6-thioguanine-resistant cells in these treated populations showed no significant increase above the background level. Comparison of the cytotoxic effect of O⁶-methyldeoxyguanosine with deoxyadenosine showed that the toxicity induced by O⁶-methyldeoxyguanosine could have resulted from mimicry of deoxyadenosine, rather than by incorporation of the alkylated nucleoside itself.     

The effect of thymidine on the induction of micronuclei by alkylating agents in V79 Chinese hamster cells

Incubation in thymidine-containing medium resulted in increased lethality and micronucleus frequency in V79 cells treated with ethyl nitrosourea (ENU), methyl nitrosourea (MNU) and ethyl methanesulphonate (EMS) but not with methyl methanesulfonate (MMS). Thymidine had no effect in ENU treated HeLa cells. In V79 cells, the presence of thymidine during post-treatment DNA replication was necessary for the effect. It is suggested that the increase in chromosome damage was the result of an increased O⁶-alkylguanine-thymine mispairing in cells which are defective in the repair of O⁶-alkylguanine. Treatment of V79 cells with O⁶-ethylguanine resulted in increased production of both micronuclei and polyploid cells. These effects might be explained by spindle dysfunction caused by the alkylated guanine.     

Inhibition of repair of O6-methyldeoxyguanosine and enhanced mutagenesis in rat-liver epithelial cells

The pro-mutagenicity of chemically-induced methylation of DNA at the O⁶ position of dexoyguanosine was studied in cultured adult rat liver epithelial cells. To modify the level of O⁶-methyldeoxyguanosine (O⁶-medGuo) resulting from exposure to an alkylating agent, partial depletion of the O⁶-alkylguanine-DNA alkyltransferase (AGT) repair system was produced by pretreatment of ARL 18 cells with a non-toxic dose of exogenous O⁶-methylguanine (O⁶-meG). Exposure of cells to 0.6 mM O⁶-meG for 4 h depleted AGT activity by about 40%. Intact and pretreated cells were exposed to a range of doses of N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), and mutagenesis at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus was quantified by measurement of 6-thioguanine-resistant mutants. The mutagenicity of MNNG was dose dependent and was greater in O⁶-meG pretreated cultures than in intact cultures. Immunoslot blot measurement of O⁶-medGuo employing a mouse monoclonal antibody demonstrated that MNNG produced O[su6-medGuo and that the intact liver cells were efficient in eliminating this lesion from their DNA. Since depletion of AGT would be expected to affect the rate of elimination of only O⁶-medGuo, it is concluded that this lesion is highly pro-mutagenic.     

Characterization of reaction products between styrene oxide and deoxynucleosides and DNA

Styrene oxide was reacted with deoxynucleosides and DNA in aqueous buffer at pH 7.4. The products were purified by HPLC, characterized by UV spectroscopy and by chemical ionization mass spectrometry. The main products identified were 7-alkyl-, N²-alkyl- and O⁶-alkyldeoxyguanosine, 1-alkyl-, and N⁶-alkyldeoxyadenosine, N⁴-alkyl-, 3-alkyl- and O²-alkyldeoxycytidine and 3-alkylthymidine. The relative yields of alkylated deoxynucleosides were dG>dC>dA>T. In the reactions of styrene oxide with DNA the dominant product isolated was 7-alkylguanine but N²-alkylguanine was also detected.     

Effect of O6-alkylguanine-DNA alkyltransferase on the frequency and spectrum of mutations induced by N-methyl-N''-nitro-N-nitrosoguanidine in the HPRT gene of diploid human fibroblasts

N-Methyl-N′-nitro-N-nitrosoguanidine (MNNG) reacts with 12 nucleophilic sites in DNA to induce a variety of lesions, but O⁶-methylguanine (O⁶-MeG) and O⁴-methylthymine are the most effective premutagenic lesions produced, mispairing with thymine and guanine, respectively. O⁶-MeG is repaired by O⁶-alkylguanine-DNA alkyltransferase (AGT), which removes the methyl group from the O⁶ position and transfers it to itself, rendering the transferase inactive. When diploid human fibroblasts were exposed to 25 μM, O⁶-benzylguanine (O⁶-BzG) in the medium for 3 h, their level of AGT activity was dramatically reduced, to a level of at most 1.6% of the control. Populations of cells pretreated with this level of O⁶-BzG for 2 h or not pretreated, were exposed to MNNG at a concentration of 2, 4 or 6 μM in the presence or absence of O⁶-BzG and assayed for survival of colony-forming ability and the frequency of 6-thioguanine-resistant cells (mutations induced in the HPRT gene). O⁶-BzG (25 μM) was also present in the appropriate half of the cells during the 24 h immediately follwing exposure to MNNG. This 27-h exposure to O⁶-BzG alone had no cytotoxic or mutagenic effect on the cells but significantly increased the cytotoxicity and mutagenecity of MNNG, increasing the mutant frequency to that found previously in human cells constitutively devoid of AGT activity. At doses of 2 μM and 4 μM MNNG, the mutant frequency observed with the AGT-depleted cells was 120 × 10⁻⁶ and 240 × 10⁻⁶, respectively; in the cells with abundant AGT activity, these values were 10 × 10⁻⁶ and 20 × 10⁻⁶, respectively. DNA-sequence analysis of the coding region of the HPRT gene in 36 independent mutants obtained from MNNG-treated AGT-depleted populations and 36 from the control populations showed that even though AGT repair lowered the frequency of mutants by more than 90%, it did not affect the kinds of mutations induced by MNNG nor the strand distribution of the premutagenic guanine lesions. In mutants from the AGT-depleted cells, there were 26 base substitutions and 13 putative splice site mutations; in the control, there were 25 base substitutions and 11 splice site mutations. All but two substitutions involved G · C with 92% being G · C → A · T. In both sets, of the premutagenic lesions were located in the nontranscribed strand. Many ‘hot spots’ were seen, and there was evidence that AGT repaired more lesions from the 5′ half of the gene than from the 3′ half.     

Why do O-alkylguanine and O-alkylthymine miscode? The relationship between the structure of DNA containing O-alkylguanine and O-alkylthymine and the mutagenic properties of these bases

The carcinogenic and mutagenic N-nitroso compounds produce GC to AT and TA to GC transition mutations because they alkylate O⁶ of guanine and O⁴ of thymine. It has been generally assumed that these mutations occur because O⁶-alkylguanine forms a stable mispair with thymine and O⁴-alkylthymine forms a mispair with guanine. Recent studies have shown that this view is mistaken and that the alkylG·T and alkylT·G mispairs are not more stable than their alkylG·C or alkylT·A counterparts. Two possible explanations based on recent structural studies are put forward to account for the miscoding. The first possibility is that the DNA polymerase might mistake O⁶-alkylguanine for adenine, and O⁴-alkylthymine for cytosine, because of the physical similarity of these bases. O⁶-Methylguanine and adenine are similarly lipophilic and X-ray crystallography of the nucleosides has shown a close similarity in bond angles and lengths between O⁶-methylguanine and adenine, and between O⁴-methylthymine and cytosine. The second possible explanation is that the important factor in the miscoding is that the alkylG·T and alkylT·G mispairs retain the Watson-Crick alignment with N1 of the purine juxtaposed to N3 of the pyrimidine while the alkylG·C and alkylT·A pairs adopt a wobble conformation. ³¹P NMR of DNA duplexes show that the phosphodiester links both 3′ and 5′ to the C have to be distorted to accomodate the O⁶-ethylguanine:C pair, whereas there is less distortion of the phosphodiesters 3′ and 5′ to the T in an ethylG·T pair. Recent kinetic measurements show that the essential aspect of base selection in DNA synthesis is the ease of formation of the phosphodiester links on both the 3′ and 5′ side of the incoming base. The Watson-Crick alignment of the alkylG·T and alkylT·G mispairs may facilitate formation of these phosphodiester links, and this alignment rather than the strength of the base pairs and the extent of hydrogen bonding between them may be the crucial factor in the miscoding. If either hypothesis is correct it suggests that previously too much emphasis has been placed on the stability of the normal pairs in the replication of DNA.     

Binding of MutS protein to oligonucleotides containing a methylated or an ethylated guanine residue, and correlation with mutation frequency

The MutS-based mismatch repair (MMR) system has been conserved from prokaryotes to humans, and plays important roles in maintaining the high fidelity of genomic DNA. MutS protein recognizes several different types of modified base pairs, including methylated guanine-containing base pairs. Here, we looked at the relationship between recognition and the effects of methylating versus ethylating agents on mutagenesis, using a MutS-deficient strain of E. coli. We find that while methylating agents induce mutations more effectively in a MutS-deficient strain than in wild-type, this genetic background does not affect mutagenicity by ethylating agents. Thus, the role of E. coli MMR with methylation-induced mutagenesis appears to be greater than ethylation-induced mutagenesis. To further understand this difference an early step of repair was examined with these alkylating agents. A comparison of binding affinities of MutS with O⁶-alkylated guanine base paired with thymine, which could lead to transition mutations, versus cytosine which could not, was tested. Moreover, we compared binding of MutS to oligoduplexes containing different base pairs; namely, O⁶-MeG:T, O⁶-MeG:C, O⁶-EtG:T, O⁶-EtG:C, G:T and G:C. Dissociation constants (K_d), which reflect the strength of binding, followed the order G:T- > O⁶-MeG:T- > O⁶-EtG:T- = O⁶-EtG:C- ≥ O⁶-MeG:C- > G:C. These results suggest that a thymine base paired with O⁶-methyl guanine is specifically recognized by MutS and therefore should be removed more efficiently than a thymine opposite O⁶-ethylated guanine. Taken together, the data suggest that in E. coli, the MMR system plays a more significant role in repair of methylation-induced lesions than those caused by ethylation.     

Properties of mer HeLa cells sensitive or resistant to the cytotoxic effects of MNU; effects on DNA synthesis and of post treatment with caffeine

A line of HeLa cells was shown to be particularly sensitive to N-methyl-N-nitrosourea (MNU) and N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), but not to variety of other cytotoxic agents. A resistant line (designated HeLa/A22), was derived by treating Hela cells repeatedly with MNU. Both the sensitive (HeLa) and resistant (Hela/A22) cells have a mer⁻ phenotype based both on their reduced rates of loss of O⁶-methylguanine (O⁶-MeG) from DNA and their low levels of the enzyme O⁶-methylguanine methyltransferase (MT). HeLa cells are therfore sensitive to unrepaired O⁶-MeG in DNA while the Hela/A22 cells are resistant to unexcised O⁶-MeG and thus the A22 cells have the mer⁻ rem⁺ phentype. MNU produced an imediate dose-dependent inhibition of DNA synthesis in cultures of both sensitive resistant cells which increased with time until about 4 h after treatment. DNA synthesis then recovered to near control rates in both sensitive and resistant cells before then exhibiting a progressive decrease after 24 h. DNA synthesis was more depressed at these late times after treatment in cultures of sensitive cells than in those of similarly-treated resistant cells. DNA synthesis remained depressed in sensitive cells but recovered 3 days after treatment in resistant cells.

Post treatment of incubation of MNU-treated HeLa cells with caffeine did not increase the toxic action of MNU. In contrast, post treatment of the resistant HeLa/A22 cells with caffeine resulted in a dramatic increase in the toxic effects of a higher equitoxic dose of MNU. The depressed rate of DNA synthesis observed in both cell lines after doses of MNU was partially reversed by post treatment with caffeine in both sensitive and resistant cells. These observations can be interpreted in terms of the effects of caffeine on DNA replication in treated cells.     

Chromosomal instability, reproductive cell death and apoptosis induced by O-methylguanine in Mex, Mex and methylation-tolerant mismatch repair compromised cells: facts and models

O⁶-Methylguanine (O⁶-MeG) is induced in DNA by methylating environmental carcinogens and various cytostatic drugs. It is repaired by O⁶-methylguanine-DNA methyltransferase (MGMT). If not repaired prior to replication, the lesion generates gene mutations and leads to cell death, sister chromatid exchanges (SCEs), chromosomal aberrations and malignant transformation. To address the question of how O⁶-MeG is transformed into genotoxic effects, isogenic Chinese hamster cell lines either not expressing MGMT (phenotypically Mex⁻), expressing MGMT (Mex⁺) or exhibiting the tolerance phenotype (Mex⁻, methylation resistant) were compared as to their clastogenic response. Mex⁻ cells were more sensitive than Mex⁺ cells to N-methyl-N′-nitro-N-nitrosoguanidine (MNNG)-induced chromosomal breakage, with marked differences in sensitivity depending on recovery time. At early recovery time, when cells out of the first post-treatment mitosis were scored, aberration frequency was about 40% reduced in Mex⁺ as compared to Mex⁻ cells. At later stages of recovery when cells out of the second post-treatment mitosis were analyzed, the frequency of aberrations increased strongly in Mex⁻ cells whereas it dropped to nearly control level in Mex⁺ cells. From this we conclude that, in the first post-treatment replication cycle of Mex⁻ cells, only a minor part of aberrations (<40%) was due to O⁶-MeG whereas, in the second post-treatment replication cycle, the major part of aberrations (>90%) was caused by the lesion. Thus, O⁶-MeG is a potent clastogenic DNA damage that needs two DNA replication cycles in order to be transformed with high efficiency into aberrations. The same holds true for sister chromatid exchanges (SCEs). MNNG is highly potent in inducing SCEs in Mex⁻ cells in the second replication cycle after alkylation. Under these conditions, SCE induction is nearly completely prevented by the expression of MGMT. This is opposed to SCE induction in the first post-treatment replication cycle, where higher doses of MNNG were required to induce SCEs and no protective effect of MGMT was observed. This indicates that SCEs induced in the first replication cycle after alkylation are due to other lesions than O⁶-MeG. In methylation tolerant cells, which are characterized by impaired G–T mismatch binding and MSH2 expression, aberration frequency induced by MNNG was weakly reduced in the first and strongly reduced in the second post-treatment mitoses, as compared to CHO wild-type cells. The results indicate that mismatch repair of O⁶-MeG–T mispairs is decisively involved in O⁶-MeG born chromosomal instability and recombination. We also show that Mex⁺ and methylation tolerant cells are more resistant than Mex⁻ cells with regard to induction of apoptosis, indicating O⁶-MeG to be also an apoptosis-inducing lesion. The data are discussed as to the mechanism of cytotoxicity, aberration and SCE formation in cells treated with a methylating agent.     

Biological significance of DNA adducts investigated by simultaneous analysis of different endpoints of genotoxicity in L5178Y mouse lymphoma cells treated with methyl methanesulfonate

The biological significance of DNA adducts is under continuous discussion because analytical developments allow determination of adducts at ever lower levels. Central questions refer to the biological consequences of adducts and to the relationship between background DNA damage and exposure-related increments. These questions were addressed by measuring the two DNA adducts 7-methylguanine (7-mG) and O⁶-methyl-2′-deoxyguanosine (O⁶-mdGuo) by LC–MS/MS in parallel to two biological endpoints of genotoxicity (comet assay and in vitro micronucleus test), using large batches of L5178Y mouse lymphoma cells treated with methyl methanesulfonate (MMS). The background level of 7-mG was 1440 adducts per 10⁹ nucleotides while O⁶-mdGuo was almost 50-fold lower (32 adducts per 10⁹ nucleotides). In the comet assay and the micronucleus test, background was in the usual range seen with smaller batches of cells (2.1% Tail DNA and 12 micronuclei-containing cells per 1000 binucleated cells, respectively). For the comparison of the four endpoints for dose-related increments above background in the low-response region we assumed linearity at low dose and used the concept of the “doubling dose”, i.e., we estimated the concentration of MMS necessary to double the background measures. Doubling doses of 4.3 and 8.7 μM MMS were deduced for 7-mG and O⁶-mdGuo, respectively. For doubling the background measures in the comet assay and the micronucleus test, 5 to 15-fold higher concentrations of MMS were necessary (45 and 66 μM, respectively). This means that the contribution of an increase in DNA methylation to biological endpoints of genotoxicity is overestimated. For xenobiotics that generate adducts without background, the difference is even more pronounced because the dose–response curve starts at zero and the limit of detection of an increase is not affected by background variation. Consequences for the question of thresholds in dose–response relationships and for the setting of tolerable exposure levels are discussed.     

Induction of lacI mutations in Big Blue Rat-2 cells treated with 1-(2-hydroxyethyl)-1-nitrosourea: a model system for the analysis of mutagenic potential of the hydroxyethyl adducts produced by 1,3-bis (2-chloroethyl)-1-nitrosourea.

We have investigated the genotoxic effects of 1-(2-hydroxyethyl)-1-nitrosourea (HENU). We have chosen this agent because of its demonstrated ability to produce N7-(2-hydroxyethyl) guanine (N7-HOEtG) and O⁶-(2-hydroxyethyl) 2′-deoxyguanosine (O⁶-HOEtdG); two of the DNA alkylation products produced by 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU). For these studies, we have used the Big Blue Rat-2 cell line that contains a lambda/lacI shuttle vector. Treatment of these cells with HENU produced a dose dependent increase in the levels of N7-HOEtG and O⁶-HOEtdG as quantified by HPLC with electrochemical detection. Treatment of Big Blue Rat-2 cells with either 0, 1 or 5 mM HENU resulted in mutation frequencies of 7.2±2.2×10⁻⁵, 45.2±2.9×10⁻⁵ and 120.3±24.4×10⁻⁵, respectively. Comparison of the mutation frequencies demonstrates that 1 and 5 mM HENU treatments have increased the mutation frequency by 6- and 16-fold, respectively. This increase in mutation frequency was statistically significant (P<0.001). Sequence analysis of HENU-induced mutations have revealed primarily G:C→A:T transitions (52%) and a significant number of A:T→T:A transversions (16%). We propose that the observed G:C→A:T transitions are produced by the DNA alkylation product O⁶-HOEtdG. These results suggest that the formation of O⁶-HOEtdG by BCNU treatment contributes to its observed mutagenic properties.     

Alkylation damage, DNA repair and mutagenesis in human cells

17 human cell lines that differ significantly in level of O⁶-alkylguanine-DNA alkyltransferase (AGT) activity were identified by comparing their sensitivity to the cytotoxic effect of N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) and determining the level of AGT activity in cell extracts from the various lines by measuring the decrease in radiolabeled O⁶-methylguanine from DNA, using high-performance liquid chromatography. 9 lines exhibited high levels of AGT activity, 2 showed an intermediate level (25–50% of the mean of those with the higher levels), and 6 exhibited very low or virtually undetectable levels of AGT. Included were several lines that are very deficient in capacity for nucleotide excision repair. When representatives from the 3 categories of cell lines defined by the level of AGT activity were compared for sensitivity to the cytotoxic and mutagenic effect of MNNG, they showed an inverse correlation between the degree of cell killing and frequency of mutants induced and the level of AGT activity. The cells' capacity for nucleotide excision repair did not affect these results. Exposure of cells with a high level of AGT activity to O⁶-methylguanine in the medium reduced the AGT activity 60–80%. These pre-treated cells exhibited a significantly higher frequency of MNNG-induced mutants than did cells that were not pre-treated, suggesting that the O⁶-methylguanine lesion in DNA is responsible for a significant proportion of the mutations induced. Cell strains containing substrates for assaying intrachromosomal homologous recombination were constructed using parental cell lines from each of the 3 categories of AGT activity. These strains showed an inverse correlation between the level of AGT activity and the frequency of MNNG-induced recombination. When various cell lines representing the 3 categories of AGT activity were compared for sensitivity to ethylnitrosourea, the results were consistent with AGT and nucleotide excision repair playing a role in preventing cell killing and mutation induction by this agent.     

Deoxynucleoside overproduction in deoxyadenosine-resistant, adenosine deaminase-deficient human histiocytic lymphoma cells

Deoxyadenosine toxicity toward lymphocytes may produce immune dysfunction in patients with adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4) deficiency. The relationship between endogenous deoxynucleoside synthesis in adenosine deaminase-deficient cells and sensitivity to adenosine and deoxyadenosine toxicity is unclear. The human histiocytic lymphoma cell line (DHL-9) naturally lacks adenosine deaminase, and has minimal levels of thymidine kinase. Dividing DHL-9 cells excrete deoxyadenosine and thymidine into the extracellular space. The present experiments have analyzed nucleoside synthesis and excretion in a mutagenized clone of DHL-9 cells, selected for increased resistance to deoxyadenosine toxicity. The deoxyadenosine-resistant cells excreted both deoxyadenosine and thymidine at a 6-7-fold higher rate than wild-type lymphoma cells. The deoxyadenosine overproduction was accompanied by a reduced ability to form dATP from exogenous deoxyadenosine, and a 2.5-fold increase in ribonucleotide reductase activity. The pace of adenosine excretion, the growth rate, and the levels of multiple other enzymes involved in deoxyadenosine and adenosine metabolism were equivalent in the two cell types. These results suggest that the excretion of deoxyadenosine and thymidine, but not adenosine, is exquisitely sensitive to alterations in the rate of endogenous deoxynucleotide synthesis. Apparently, small changes in deoxynucleotide synthesis can significantly influence cellular sensitivity to deoxyadenosine toxicity.     

Quantitative comparison of carcinogenicity, mutagenicity and electrophilicity of 10 direct-acting alkylating agents and of the initial O6:7-alkylguanine ratio in DNA with carcinogenic potency in rodents

The quantitative relationship between carcinogenicity in rodents and mutagenicity in Salmonella typhimurium was examined, by using 10 monofunctional alkylating agents, including N-nitrosamides, alkyl methanesulfonates, epoxides, β-propiolactone and 1,3-propane sultone. The compounds were assayed for mutagenicity in two S. typhimurium strains (TA1535 and TA100) and in plate and liquid assays. The mutagenic activity of the agents was compared with their alkylating activity towards 4-(4′-nitrobenzyl)pyridine and with their half-lives (solvolysis constants) in an aqueous medium. No correlations between these variables were found, nor was mutagenic activity correlated with estimates of carcinogenicity in rodents.

There was a positive relationship between carcinogenicity and the initial ratios of 7-: O⁶-alkylguanine formed or expected after their reaction with double-stranded DNA in vitro. The results suggest that alkylation of guanine at position O⁶ (or at other O atoms of DNA bases) may be a critical DNA-base modification that determines the overall carcinogenicity of these alkylating agents in rodents.     

Induction of rat liver O6-alkylguanine-DNA alkyltransferase by bleomycin

Alkyl adducts at the O⁶-position of guanine constitute promutagenic DNA lesions likely to be involved in the initiation of malignant transformation. They can be removed by a cellular acceptor protein termed O⁶-alkylguanine-DNA alkyltransferase (AT). In rat liver this repair enzyme can be induced by a variety of hepatotoxins, partial hepatectomy and X-irradiation. This paper describes a stimulation of the hepatic AT by treatment of rats with the radiomimetic agent, bleomycin. Induction of AT is dose-dependent up to 20 mg bleomycin/kg and appears to level off with higher doses. Enhancement of O⁶-meG repair is detectable within 24 h after a single i.p. injection. Maximum AT induction was reached after 6 days and amounted to 350% of the control levels. The enhancement of AT activity is not associated with acute liver injury and initially coincides with an inhibition of [³H]deoxythymidine incorporation into hepatic DNA. This indicates that AT induction in rat liver is not necessarily dependent on tissue necrosis with increased cell replication. Since bleomycin does not produce DNA lesions recognized and repaired by the AT, the hypothesis is entertained that AT induction by these agents is part of a concerted reaction to radiation-type DNA damage.     

Specificity of nucleotide binding and coupled reactions utilising the mitochondrial ATPase

1. 1. Tightly bound ATP and ADP, found on the isolated mitochondrial ATPase, exchange only slowly at pH 8, but the exchange is increased as the pH is reduced. At pH 5.5, more than 60% of the bound nucleotide exchanges within 2.5 min.

2. 2. Preincubation of the isolated ATPase with ADP leads to about 50% inhibition of ATP hydrolysis when the enzyme is subsequently assayed in the absence of free ADP. This effect, which is reversed by preincubation with ATP, is absent on the membrane-bound ATPase. This inhibition seems to involve the replacement of tightly bound ATP by ADP.

3. 3. Using these two findings, the binding specificity of the tight nucleotide binding sites was determined. iso-Guanosine, 2′-deoxyadenosine and formycin nucleotides displaced ATP from the tight binding sites, while all other nucleotides tested did not. The specificities of the tight sites of the isolated and membrane-bound ATPase were similar, and higher than that of the hydrolytic site.

4. 4. The nucleotide specificities of ‘coupled processes’ nucleoside triphosphate-driven reversal of electron transfer, nucleoside triphosphate-³²P_i exchange and phosphorylation were higher than that of the hydrolytic site of the ATPase and similar to that of the tight nucleotide binding sites.

5. 5. The different nucleotide specificities of uncoupled ATP hydrolysis and coupled processes can be explained even if both processes involve a single common site on the ATPase molecule. This model requires that energy can be ‘coupled’ only when it is released/utilised in the nucleotide binding steps of the mechanism.

6. 6. Adenosine β,γ-imidotriphosphate (AMP-PNP) is not a simple reversible inhibitor of the ATPase, since incubation requires preincubation and is not reversed when the compound is diluted out, or by addition of ATP. This compound inhibits the isolated and membrane-bound ATPase equally well. Its guanosine analogue does not act in this way.

7. 7. In submitochondrial particles, ADP inhibited uncoupled hydrolysis of ATP much more effectively than coupled hydrolysis, the latter being measured both directly (from ATP hydrolysis in the absence of uncoupler) or indirectly, by monitoring ATP-driven reduction of NAD⁺ by succinate.

8. 8. The effects of ADP and AMP-PNP were interpreted as providing evidence for two of the intermediates in the proposed scheme for coupled triphosphate hydrolysis.

Dynamics of the dATP pool in cultured mammalian cells.

Conditions for labeling the dATP pool of V79 and 3T3 cells from [3H]deoxyadenosine (salvage) or [3H]adenine (via ribonucleotide reduction) were established. With deoxyadenosine the specific radioactivity of dATP reached a constant value after 60 min. In resting 3T3 cells this value was 30 times higher than in S-phase cells. Turnover of dATP and absolute rates of DNA synthesis and excretion of breakdown products of dATP were determined from the accumulation of isotope in various compartments and the specific activity of dATP. In S-phase cells the dATP pool had a half-life of 4 min, identical to that of dTTP determined earlier. Deoxyadenosine was the major breakdown product of dATP in the presence of an inhibitor of adenosine deaminase. The rate of deoxyadenosine excretion of V79 cells amounted to 4% of the rate of dATP incorporation into DNA. Inhibition of DNA replication increased deoxyadenosine excretion 5- to 10-fold, demonstrating a continued de novo synthesis of dATP, albeit at a slightly reduced rate. Our results fit a model involving a substrate cycle between dAMP and deoxyadenosine regulating the dATP pool, similar to the model of substrate cycles involved in the regulation of pyrimidine deoxyribonucleotide pools developed earlier.     

Specific selection of deoxycytidine kinase mutants with tritiated deoxyadenosine

We have shown previously that a low concentration of tritiated deoxyadenosine, i.e., 1 µCi/ml, selectively kills wild-type S49 murine lymphoma cells. Mutant cells resistant to [³H]deoxyadenosine lacked adenosine kinase completely but retained a significant level of deoxyadenosine phosphorylating activity. To study further the specificity of [³H]deoxyadenosine selection, lymphoma cell clones resistant to 15 µCi/ml [³H]deoxyadenosine have been derived. The resistant line, S49-dA15, is also resistant to high levels of nonradioactive deoxyadenosine and to deoxyguanosine but remains sensitive to thymidine. The thymidine inhibition of the growth of the mutant, in contrast to that of the wild-type cells, cannot be prevented by deoxycytidine. The mutant line lacks deoxycytidine kinase that also phosphorylates deoxyadenosine. In addition, the mutant cells excrete a large amount of deoxycytidine into culture medium, consistent with a failure of salvage of the nucleoside in the absence of an appropriate kinase, i.e., deoxycytidine kinase. In contrast, a deoxycytidine kinase-deficient cell line that was selected with arabinosylcytosine does not excrete deoxycytidine and contains high deoxycytidine deaminase activity. [³H]Deoxyadenosine can be used as a selective agent for specific selection of deoxycytidine kinase-negative mutants.     

Effect of adenosine deaminase inhibitors on the apparent rate of phosphorylation of deoxyadenosine

The apparent rate of phosphorylation of deoxyadenosine has been studied in crude extracts of human leukemic cells. Since detection and quantitation of phosphorylated products of deoxyadenosine are not possible in the presence of the competing enzyme, adenosine deaminase, an assay system has been devised in which deaminase activity is totally inhibited. Two inhibitors of adenosine deaminase, erythro-9-(2-hydroxy-3-nonyl)adenine and 2′-deoxycoformycin were tested. Complete inhibition of adenosine deaminase cannot be achieved with erythro-9-(2-hydroxy-3-nonyl)adenine, but can be achieved with 2′-deoxycoformycin at concentrations greater than 100 μ m. Use of deoxycoformycin allows an accurate assessment of phosphorylation of deoxyadenosine and its nucleoside analogs. Cellular extracts from patients with several types of leukemia contained a 30-fold difference in relative deoxyadenosine kinase activity. Adenine arabinoside is a competitive inhibitor of deoxyadenosine, but not adenosine phosphorylation with a K_{i (app)} of 3.2 mm. This inhibition pattern is consistent with a common pathway of phosphorylation for deoxyadenosine and adenine arabinoside in human leukemic cells.