Elsevier

Biochimie

Volume 82, Issue 8, August 2000, Pages 717-721
Biochimie

Modified alkaline elution allows the measurement ofintact apurinic sites in mammalian genomic DNA

https://doi.org/10.1016/S0300-9084(00)01152-4Get rights and content

Abstract

The presence of apurinic/apyrimidinic (AP) sites in cell genomes is known to be toxic and mutagenic. These lesions are therefore repaired in cells by efficient enzymatic systems. However, a report (Nakamura and Swenberg, Cancer Res. 59 (1999) 2522–2526) indicates an unexpected high rate of endogenous apurinic/apyrimidinic (AP) sites in genomic DNA in mammalian tissues. The technology used does not allow the authors to distinguish between intact AP sites and 3’cleaved AP sites. The corresponding values range between 2 and 4 sites per million of nucleotides in various human and rat tissues. Using a modified alkaline elution method we show here that the stationary level of intact AP sites is about 0.16 per million of nucleotides in leukemic mouse L1210 cells.

Introduction

The glycosidic bond between bases and deoxyribose in DNA is labile under certain conditions such as heating, alkylation of bases or the action of N-glycosylases [1]. The cleavage of the glycosidic bond in DNA leads to an abasic site which is chemically characterized for the deoxyribose by an equilibrium between a closed ring and an aldehydic open one. The reaction of this aldehyde with hydroxyle ions [2] or primary amines [3] leads to a primary DNA cleavage in 3’ through beta elimination and then to secondary cleavage in 5’ through delta elimination which leads to a complete removal of the deoxyribose ring from the DNA molecule.

The high frequency of induction of AP sites in the genome of both bacteria and mammalian cells by physical and chemical agents is a well documented phenomenon [4]. These non-coding lesions have been shown to be mutagenic in both prokaryotes and eukaryotes [5], [6]. The loss of purines takes place at a twenty times higher rate when compared to pyrimidines [7]. The rate of spontaneous purine release at 37 °C, pH 7.4, has been measured in vitro on bacterial DNA [8] and the corresponding estimation for the depurination events during a cell generation is 0.3–1.5 per million of nucleotides. More recently this rate has been confirmed at 1.35 sites per million of nucleotides [9]. However, enzymes that repair abasic sites are present in all biological systems studied [10]. Taking into account the efficiency of these repair enzymes the stationary level of abasic sites in cells was supposed to be very low. However, a surprisingly high level of intact abasic sites had already been found in the PM2 phage DNA extracted from Pseudomonas aeruginosa bacteria [11]. An article published in 1999 in Cancer Res. [12] shows very unexpectedly that there are 8.3 to 33.3 abasic sites per million of nucleotides in vivo. This report confirms a previous report by the same team indicating that ex vivo cells have 3.8 sites per million of nucleotides in the genome [2]. This had not been characterized before because of technical problems related to sensitivity and specificity of existing methods. The authors divide these sites in 5’ cleaved, 3’ cleaved + intact sites and residual sites which are uncleaved aldehydic lesions. It is important to evaluate the frequency of the intact sites which might escape repair and therefore be more mutagenic. Several methods are available to measure AP sites. These methods include methoxyamine which reacts with the aldehyde group [13], antibodies against O-4-nitrobenzylhydroxylamine bound to abasic sites [14], 5’ 32[P]-postlabeling [15] and the use of the aldehyde reactive probe (ARP) [16]. However, most of these methods are unable to directly measure 3’ cleaved AP sites because the reagent does not distinguish cleaved from intact AP sites. To measure 3’ cleaved AP sites the method using ARP requires 5’ cleavage with exonuclease III but unspecific cleavage also occurs in 3’ [12] making the quantification impossible. 3’cleavage triggered by alkali or a primary amine is therefore a very good way of measuring intact sites through the number of DNA breaks. Accordingly we have used the properties of cleaved DNA to perform the measurement of these intact sites in cultured mouse leukemic L1210 cells. The measurements of these intact cellular abasic sites by DNA cleavage are generally performed by the two pH alkaline elution method (pH 12.1 and pH 12.6). However, this is a moderatly sensitive, non-quantitative assay because cleavage taking place during elution results in concave elution curves. Under these experimental conditions cells which have not been submitted to any treatment show very low levels of abasic sites in their DNA. Here we have used an improved alkaline elution method that allows the determination of the basal level of abasic sites in the absence of DNA repair.

Section snippets

Cell and radioactive labeling

L1210 mouse leukemia cells were grown in non-agitated suspension culture in RPMI 1640 medium supplemented with 10% fetal calf serum, 2 mM glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin. In this medium, cells (doubling time of 12–13 h) were grown exponentially up to 1.5 × 106 cells/mL in a saturated humidity with 5% CO2 at 37 °C.

Cellular DNA in exponentially growing cells (2 × 105 cells/mL) was radioactively labeled by incubation with [2-14C] thymidine (0.74 kBq/mL, 1.9 GBq/mmol,

Results

In the new assay the alkaline elution technique [2] is coupled with a preincubation of cellular DNA at pH 7 in the presence of the tripeptide lysyl-tryptophyl-α-lysine (Lys-Trp-Lys) which is known to recognize and cleave DNA at abasic sites [18], [19], [20]. DNA from L1210 cells was isolated on polycarbonate filters. The incubation with Lys-Trp-Lys has been performed after cell lysis to avoid any problem of tripeptide penetration or of AP site repair. SSB have been then measured by alkaline

Discussion

If we estimate the rate constant for spontaneous depurination from the values given by [8] we find that during the 30-min incubation period 0.018 AP sites per million of nucleotides are formed in the whole genome. The difference is therefore around 0.16 sites per million of nucleotides in L1210 cells. This has to be compared to 2 to 4 intact + 3’ cleaved sites per million of nucleotides measured by Nakamura and Swenberg [12]. We can therefore hypothesize that there is only a minority of intact

Acknowledgements

This work was supported by the grant 210792 from ARC: ‘Potentialisation d’agents anticancéreux par inhibition de la réparation par excision de base’. Marc Lefrançois was supported by the Institut de Formation Supérieure Biomédicale.

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