DNA single-strand breaks in adult and embryonic avian erythrocytes

EXPERIMENTAL

CELL

RESEARCH

190,141-144

(1990)

SHORT NOT A Single-Strand Breaks in Adult and Embr S. P. GUY,* T. K. BRADSHAW,~

AND Ft. F. ITZHAKI*,~

*Molecular Neurobiology Laboratory, Department of Optometry and Vision Sciences, University of Manchester Institute of Science am-j Technology, Manchester M60 1QD; and TShell Research, Ltd., Sittingbourne Research Centre, Sittingbourne, Kent ME9 BAG, United Kingdom

The life span of adult erythrocytes is greater than that of embryonic cells, the former being 31 days in the blood and the latter, for the P2-day “definitive” embryonic cells we use, 6 days or less (““definitive” cells begin appearing in the blood at 6-7 ays ~~stfertilization). Several studies have indicated that with increasing age of the cell or organism, or with degree there is an accumulation of lesions in lar, an increase in the number of sin (ssb) [4-71; this accumulation has been attributed to a decrease in activity of enzymes involveci in repair of endogenous damage. Consistent with this, little or no repair of DNA occurs after exogenou amage such as uv irradiation [8], X-irradiation [9] 9or NU treatment [I, tio~ rate of the 21. On this basis, the slower reac adult compared to that of the embrycmic cell could reflect a greater number of ssb, e to the difference in their ages and/or possibly to a rence in activity of their repair enzymes, if present. We describe here a comparison of ssb in the DNA of adult and embryonic erythrocytes. As a measure of the number of breaks in the DNA, we have assayed the template activity of chromatin in isola added DNA polymerase I [S, 10-E]. such as alkaline sucrose gradient ten kaline elution, which give a measure of ssb (but which assay also alkali-labile sites), coul not be used since they are valid only if the number s used is small; hence, for detection purposes, the must be radiolabeled, but labeled DNA cannot be ob~a~~ed from avian erythrocytes since they do not synthesize any DNA.

We have investigated the possibility that the reactivation rate of adult avian erythrocytes, which is slower than that of embryonic erythrocytes, after fusion with metabolically active cells, is due to a greater number of single-strand breaks (ssb) in the DNA of the former. We have assayed ssb by measuring the template activity of the erythrocyte nuclei for added Escherichia coli DNA polymerase. We have found that differences in the numbers of ssb within polymerase-accessible regions between adult and embryonic eells are within experimental error. We conclude that, unless very localized clusters of damage exist within the DNA (which would not be detectable by this or other techniques), the difference in reactivation rate is not attributable to differences in ssb numbers. 0 1990 Academic Press, Inc.

INTRODUCTION

Avian erythrocytes are terminally differentiated cells; they contain highly condensed chromatin and synthesize no DNA or proteins and only minimal levels of RNA. Fusion of these cells with metabolically active cells leads to reactivation of the erythrocyte nuclei, the initial event being a swelling of the nuclei followed by reappearance of nucleoli and, eventually, synthesis of avian-specific proteins. We have examined reactivation of erythrocytes in a study of the effects of an alkylating agent, N-methyl-N-nitrosourea (MNU), on these cells [lt 21. Our measurements of rates of nuclear swelling and of reappearance of nucleoli have shown that reactivation of erythrocyte nuclei from adult hens is slower than that of embryonic erythrocyte nuclei, in agreement with the studies of Harris and co-workers (see, e.g., Ref. [3]). The reason for this difference is unknown, but one possibility is that the DNA of adult cells is more damaged than that of embryonic cells.

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Addt erythrocytes were Preparation of erythrocytes and nuclei. prepared as described previously [l] from Rhode Island Red X White Leghorn hens, aged 18-24 months. Embryonic erythrocytes were obtained from fertilized hens’ eggs incubated at 38°C for 12-14 days. Nuclei were prepared by lysis of the cells in reticulocyte standard buffer (RSB)-10 mMTri-HCl, pH 7.4,lO mM NaC1,3 mMMgCl,, containing 0.5% Nonidet-P4Q. The nuclei were tken washed several times in RSB (without Nonidet) to remove hemoglobin.

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141

0014-4527/W $3.00 Copyright

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142

SHORT

TABLE Effect

of DNAase Erythrocyte

(pglml) 0 1 2 3

TABLE

1

I on Template Activity of Adult Nuclei for DNA Polymerase Incorporation

DNAase

Expt lb 88 645 973 1570

NOTE

+ 2 f t

0.2 3 4 5

of dCTP”

92 505 695 1536

Template Activity of Fresh and Stored Avian Erythrocyte Nuclei

Avian

Incorporation

(fmol)

Expt 2” rf: t f f

0.2 4 4 10

a Amount of DNA per assay was 50 pg; incubation was for 30 min at 37°C. b Mean and standard deviation for two or three aliquots.

Assay of template activity. Samples were set up in duplicate or triplicate. Nuclei were suspended in 0.01 M Tris-HCl, pH 7.4, 0.3 M sucrose, 1 mM ,B-mercaptoethanol. Aliquots containing 50 pg DNA (taking the DNA content of the erythrocyte to be 3 pg) were mixed with dGTP, dATP, and dTTP (final concentrations 25 PM), 1 PCi [5-3H]dCTP (26.3 Ci/mmol) and 5 units Escherichia coli DNA polymerase I, in 0.05 MTris-HCl, pH 7.6,7 mMMgCl,, 1 mM dithiothreitol, 4 mM ATP (total volume 100 ~1). Samples were incubated for 30 min. The incubation temperature was usually 37°C but some of the assays were done at 16°C so as to preclude any “strand displacement,” i.e., net synthesis of DNA, which can occur at temperatures above about 22°C. In some experiments, calf thymus DNA (0.1 pg) was assayed for comparison. Radioactivity incorporated into DNA was measured by precipitation of the nuclei with ice-cold 0.5 M perchloric acid (PCA)-4% sodium pyrophosphate, for 1 h. Unincorporated radioactivity was removed by centrifugation of the acidified nuclei at 12,000g for 30 s followed by repeated washing of the pellet with PCA until the counts in the supernatant reached background level. To solubilize the DNA, the final nuclear pellet was heated in 1 ml 0.5 M PCA at 90°C for 30 min; the mixture was then centrifuged at 12,000g and the supernatant counted in 4.5-ml triton-toluene phosphor.

RESULTS

We found the agreement between duplicate or triplicate assays of incorporation for any one sample to be excellent (Table 1); however, there was sometimes considerable variability between different samples, i.e., different preparations of nuclei. This could be due either to slight differences in extent of handling of different samples, or possibly to differences in action of any endogenous nucleases; it may be relevant that chick erythrocyte nuclei have been found to contain DNA-relaxing enzyme [ 131, the effect of which could vary between samples. In all cases, the amount of radioactivity incorporated was vastly less than that incorporated into extracted calf thymus DNA, presumably the latter incurs a high degree of damage during isolation from tissue. To check that DNA polymerase was present in excess in the assay, DNAase I was added at a range of concentrations to the incubation mixture so as to introduce additional ssb into the DNA. Table 1 shows that incorporation increased with increasing levels of DNAase and in all cases was far greater than in the sample containing no DNAase; this indicates that the amount of

2

Sample Adult nuclei (fresh) Adult nuclei (1 week old) Embryonic nuclei (fresh) Embryonic nuclei (1 week old) Calf thymus DNA

of dCTP (fmol)”

Expt 1”

Expt 2b

34*

1

40*

1

182

1

26 rt

0.4

34 rt

0.4

54 2

0.1

20 k

0.2

34 *

0.4

40 *

0.2

35*

1

1255 + 17

1205 + 19

Expt 3”

1388 f 20

DAmount of DNA per assay was 50 pg for nuclei, 0.1 pg for calf thymus DNA; incubation was for 30 min at 37°C. b Mean and standard deviation for two or three aliquots.

polymerase used was adequate for assaying ssb present in undigested nuclei. An experiment with DNAase added to embryonic nuclei (at 16°C) gave a similar result (not shown). To find whether stored cells could be used for preparation and assay of ssb, nuclei prepared from fresh blood were compared with those prepared from blood maintained for a week (diluted 1:l in Alsever’s solution) at 4°C. Table 2 shows that values of incorporation into the two sets of nuclei were not significantly different for the embryonic samples although they decreased (surprisingly, since, if anything, an increase might be expected) in the adult samples. Table 3 compares incorporation into adult and embryonic cells in three further preparations. Clearly there is no significant difference between the two types of nuclei. The incorporation after various incubation periods was examined in order to find if the shapes of the time curves were similar for the two types of nuclei, i.e., if their incorporation after any particular time period

TABLE

3

Template Activity of Adult and Embryonic Avian

Erythrocytes Incorporation

of dCTP (fmol)”

Sample

Expt lb

Expt 2b

Expt 3b

Adult nuclei Embryonic nuclei Calf thymus DNA

57f 1 38 f 0.6 1081 -c 11

36 -t 0.7 45 -t 0.5 780 f 12

25 t- 0.9 18 zk 0.4

DAmount of DNA per assay was 50 pg for nuclei, 0.1 pg for calf thymus DNA; incubation was for 30 min at 37°C. b Mean and standard deviation for three aliquots.

SHORT

TABLE

4

Template Activity of Avian Erythrocyte Nuclei after Different Incubation Periods Sample of nuclei

Time (min)

Incorporation of dCTP” (fmol)

Add

10 20 30

14 + lb 11 + 2 10 + 3

Embryonic

10 20 30

18? 1 19 * 1 21 f 3

a Amount of DNA per assay was 50 fig; incubation was for 30 min at 16°C. * Mean and standard deviation for two or three aliquots.

could be directly compared. Table 4 shows that the incorporation (at 16°C) in both adult and embryonic nuclei was complete within 10 min and indicates again that differences in incorporation for the two types of nuclei were within experimental error. DISCXJSSION

Our results show that the template activities of adult and embryonic nuclei are similar. Our previous studies have indicated that accessibility of chromatin to DNA polymerase is restricted, compared to that of DNA [lo, 121. If the accessibility differed between adult and embryonic erythrocytes, our present findings would be explicable only if such a difference were balanced by a compensating difference in number of ssb within the polymerase-accessible regions, and this seems very unlikely. Thus the numbers of ssb are probably similar in the adult and embryonic cell; they are also extremely low compared to that of other cell types, the template activity being about BOOO-fold less than that of rat cardiac muscle nuclei [4] or of chromatin from rat thymus ]lO], rat liver [ll], and Ehrlich ascites cells [6]. This is unlikely to be due to a lower accessibility of DNA reflecting the extreme condensation of the avian nuclei: the ehromatin and cardiac muscle studies showed that template activity increases (although merely 2- or 3fold) rather than decreases with extent of condensation or degree of differentiation [4], or with age [6], the cause being an increase in ssb, not in accessibility. Further, the template activity differs only about 3-fold or less between chromatin from rat thymus, rat liver, and ascites cells, which differ in their extent of condensation. Our suggestion that avian erythrocyte DNA has relatively few ssb is in accord with the study of Mandal ed al. 1141; the latter proposed, on the basis of viscosity and sedimentation measurements, that avian erythrocyte DNA was far less degraded than calf thymus and chick liver DNA (although extracted DNA that was probably appreciably damaged during isolation was USd).

143

NOTE

In our cells, the difference in age of adult and embryonic cells, or in activity of any repair enzymes, may be too small to cause a detectable difference in numbers of ssb. Alternatively, the extreme ~ete~o~I~romatizatio~ of both types of nuclei may preclude appreciable endogenous damage in the DNA. The only previous relevant report on avian erythrocytes (adult cells) maintained that ssb (assayed with alkali-labile sites) increased with increasing age of the cells [9]; on this basis, our adult DNA should have more ssb than ernb~~~~~~~DNA. ever, the type of “young” cell axarn~~e~ by Karra Qrmerod [9] is uncertain: the c were obtained by repeatedly bleeding a hen by c c puncture, thereby reducing the number of matu s and increasing the number of erythroid precursors in the circulation. We bave found tbat acces micrococcal nuelease differs between adult initial rate of DNA digestion of ad than that of embryonic nuclei [B5]. Since micrococcal nuclease (P6,800 Da) is much smaller tha PQlYmerase (109,000 Da), accessibility of the tin to the nuclease is probably greater than to the polymerase; thus the different digestion rates presumably reflect differences in fine structure between a onic chromatin, i.e., at a level inacces merase. Consistent with this, in t chromatin of the inactive X chromoso ity to DNA polymerase but not to D is restricted. Our technique for assaying ssb, like alkaline gradient centrifugation and alkaline elution, does not detect any very localized clusters of ssb. If these existed in one but not the other type of erythroeyte, in regions especially reactivation process, it could ac in reactivation rates between 1s. It may be relevant that differences have been detectecl between adult and U-day embronic erythrocytes in rnmleoid se~rne~t~t~~~ rate, possibly due to a different degree of eonst in the DNA [IT]. ithin DNA accesWe conclude that the numbers of s sible to DNA polymerase, in the nuclei of day embryonic erythrocytes do not differ therefore unless a difference in number o the cell types ex s within specific localized regions of the genome, the renee in their ~~a~~iv~t~~~ rates is probably unrelated to numbers of strand breaks. This work was supported with Shell Research, Ltd.

by an SERC CASE award in conjunction

EFERENCES 1. 2. 3.

Ward, T. II., and Itzhaki, R. I?. (1981) J. Cell Sci. 51, 153-162. Guy, S. P., Bradshaw, T. K., and Itzhaki, R. P. (1990) Chem. Biol. Interact. ‘74, 207-220. Harris, IX, Sidebottom, E., Grace, D. M., and Bramwehl, &‘I. E. (1969) J. Cell Sci. 4,499-525.

144 4. 5. 6. 7. 8. 9. 10. 11.

SHORT Claycomb, W. C. (1978) Biochem. J. 171,289-298. Farzaneh, F., Zalin, R., Brill, D., and Shall, S. (1982) Nature (London) 300,- 362-366. Baghdjian, R. B., Itzhaki, S., Ockey, C. H., and Itzhaki, R. F. (1982) Mol. Cell. Biochem. 49, 169-175. Vijg, J., and Knook, D. L. (1987) Geriatr. Biosci. 35, 532-541. Darzynkiewicz, Z. (1971) Exp. Cell Res. 69, 477-481. Karran, J., and Ormerod, M. G. (1973) Biochim. Biophys. Actu 299,54-64. Itzhaki, R. F., and Safiill, R. (1973) Eur. J. Biochem. 35,259265. Saffhill, R., Cooper, H. K., and Itzhaki, R. F. (1974) Nature (Lordon) 248,153-156.

Received February 28,199O Revised version received May 11, 1990

NOTE 12.

Sat&ill, 119.

13.

Bina-Stein, M., Vogel, T., Singer, D. S., and Singer, M. F. (1976) J. Biol. Chem. 251,7363-7366.

14.

Mandal, R. K., Mazumder, H. K., and Biswas, B. B. (1974) in Control of Transcription (B&was, B. B., Mandal, R. K., Stevens, A., and Cohan, W. E., Eds.), Basic Life Sci., Vol. 3, pp. 295-302, Plenum, New York.

15.

Guy, S. P., Bradshaw, T. K., and Itzhaki, Int. Rep. 13,759-771.

16.

Dyer, K. A., Riley, D., and Gartler, S. M. (1985) Chromosoma 92,209-213. Cook, P. R., and Brazell, I. A. (1976) J. Cell Sci. 22, 287-302.

17.

R., and Itzhaki,

R. F. (1975) Nucleic Acids Res. 2, 113-

R. F. (1989) Cell Biol.