Int. _I. Radicltion Oncology Biol. Phys..

1976, Vol.

1, pp. 289-W.

Pergamon Press.

Printed in the U.S.A.

METHYLATION OF DNA IN HeLa CELLS mER ULTRAVIOLET IRRADIATION? MARGARET

Low, B.Sc., PH.D., EVELYN

L. READ

and ERNEST BOREK, PH.D. Department

of Microbiology, University of Colorado Medical Center, Avenue, Denver, CO 80220, U.S.A.

4200East Ninth

The synthesii of DNA and its methylation were measured in HeLa cells after exposure to increasing doses of ultraviolet (UV). The extent of methylation relative to DNA synthesis increases in a dose dependent manner. “Old” and “new” DNA were separated by gradient centrihgation and the effect of UV on the methylation of the two species was deter&ted. Methyfation of %ew” DNA is a&c&d more extensively than the methytation of “old” DNA by exposure to irradiation. The possible significance of aberrant methylation of DNA in potentiating UV induced damage is discussed. Methylation of DNA, HeLa cells, Ultraviolet irradiation, Isotopes.

INTRODUCTION Several possible

functions

of the methylated

bases of DNA have been suggested, all of which involve control, the methyl groups serving as signals for interaction with appropriate enzymes. These are, DNA replication,‘o*22 nuclease activity,5,‘6 and transcription.“‘” A small fraction of the methylated bases of DNA has been established as playing an important role in recognition in modification restriction systems in bacteria.6*2’ One process which involves several enzymes of DNA metabolism is excision repair. Human cells, like bacteria, require this multicomponent system as a vital part of their recovery from UV-irradiation damage. The steps involved are thought to be an initial incision near a lesion in the DNA; the removal of nucleotides from the damaged strand; resynthesis of the excised region; and the sealing of this new piece into the preexisting

It is not established what role, if any, the methylated bases play in irradiation damage or recovery therefrom in the process of excision repair. Studies of the methylation of DNA after UV-irradiation of E. coli have revealed aberrant patterns of methylation.‘**‘9*u We report here studies of DNA synthesis and methylation in HeLa cells, a cell line of human origin, after UV-irradiation.

METHODS

AND MATERIALS

DNA.‘.”

Medium and isotopes Minimal essential medium and fetal calf serum were obtained from Grand Island Biological Company. The isotopes used were [methyl-“Clthymidine, specific activity 54.5 mCi/mmole. New England Nuclear Corp, [2-?I-Cytidine, specific activity 29 mCi/mmole, Schwartz/ Mann; Y-deoxyadenosine, specific activity 447 mCi/mmole; [methyL3H]-L-Methionine,

+This work was supported by Contract AT (1 l-l) 2066 from the U.S. Atomic Energy Commission. Part of this work was presented at a Symposium organized by the Federation of European Biochemists, Budapest, Hungary (August 1974).

Abbreviations used: UV, ultraviolet; SSC, dard saline citrate; TCA, trichloroacetic d’ITP, deoxythymidine triphosphate; sp. specific activity; O.D., optical density: dpm, tegrations per minute. 289

stanacid; act., disin-

290

RadiationOncology0

Biology 0 Physics

specific activity 2 Cilmmole Schwartz-Mann. Ribonuclease was purchased from Worthington Biochemical Corp., and pronase, B grade and 5-bromodeoxyuridine (BrUdR) from Calbiochem. Cell culture HeLa cells (from American Type Tissue Culture) were grown routinely in minimal essential medium supplemented with 10% fetal calf serum, on Falcon plastic 100 mm petri dishes, at 37”in a humidified atmosphere with 5% CO* in air. Minimal essential medium containing methionine reduced from 100 to 33 pmolar, and supplemented with 10 mmolarsodium formate was used for labelling with [methyl-‘HI-L-methionine. Isotopes were used at specific activities of l%O~Ci/~mole and 0.3-O-8 [methyl-3H]-L-methionine, p Ci/p mole [methyl-“Cl-thymidine, [‘“Clcytidine, or “C-deoxyadenosine. These DNA precursors were 5 pmolar in the medium. For density-labelling of DNA, cells were incubated in the presence of Fmolarbromodeoxyuridine. W-irradiation

The medium was aspirated from exponentially growing monolayers of HeLa cells, which were then washed with phosphate buffered saline, Dulbecco’s modification (PBS). After aspiration, the cells were UVirradiated with a germicidal lamp at a doserate of 5 ergs/mm2/sec. Cells were then incubated in isotope-containing medium as described above, and in the Legends.

Vol. I, No. 34

standard saline citrate (SSC) for further purification by repetition of the above steps and, finally by incubating for 18 hr at 37” in 0.4 M-NaOH. The remaining TCA-insoluble material was washed thoroughly with 5% (w/v) TCA, ethanol and ether.

CsCl gradient analysis To investigate the distribution of methyl groups in DNA, cells were prelabelled for 48 hr before irradiation with a ‘“C-labelled DNA precursor. After irradiation of O5OOergslmm’ they were incubated with [methyl-‘HI-L-methionine and 10” M bromodeoxyuridine for 4 hr. Parallel cultures were not exposed to pre-labelling; instead, after the UV irradiation they were incubated with both labels simultaneously for 4 hr. DNA was isolated as described above. Solutions of DNA in SSC were dialyzed against T.N.E. (O-01M-tris-HCl, pH 8.0, O-01M-NaCl, 0.05 M-EDTA). The DNA was then banded by centrifugation in CsCl, as described by Edenberg and Hanawalt! Seven-drop fractions were collected from the bottom of the tubes, were diluted with 0.5 ml water, and the optical density was measured. NaOH solution was added to the samples to 0.4M. Carrier DNA was added and the mixture was incubated at 37” for 18 hr to degrade any remaining RNA. The trichloroacetic acid (TCA)-insoluble precipitates were collected on glass fibre discs, washed thoroughly with 5% (w/v) TCA and ethanol, and radioactivity trapped on the discs was measured in a toluene-based liquid scintillation fluid.

Isolation of DNA

After isotopic labelling, the cell cultures were washed repeatedly with PBS and were then scraped into PBS. They were pelleted at 1OOOg for 5 min and suspended in 0.15M-NaCl, 0.015 M-trisodium citrate, pH 7-O (SSC) and incubated at 37” for 60min with pronase (200 pg/ml, previously self-digested at 37”, for 45 min) RNAase (40 &ml, DNAase-free) and sodium dodecylsulphate (0*4%, w/v). DNA was extracted by the method of Marmur” and either precipitated in 70% ethanol or used directly for CsCl density gradient analysis. The precipitated DNA was redissolved in

DNA base analysis

DNA purified directly from cells or alkalidigested and precipitated from pooled fractions from CsCl gradients was hydrolyzed in 88% formic acid at 175°C for 60 min in sealed tubes. The dessicated hydrolysate was dissolved, 5 meCyt was added, and aliquots were applied to cellulose thin layer plates (from Eastman-Kodak). Two-dimensional chromatography was performed using, as solvents, n-butanol; H20; ammonia (86: 10: 4, v/v/v) in the first direction, and iso-propanol; cont. HCl; H20 (68: 16.2: 15-8, v/v/v) in the second.

Methylation

of DNA in HeLa cells after ultraviolet irradiation 0 M.

RESULTS DNA methyiation relative to synthesis Table 1 summarizes the results of DNA base analyses comparing DNA methylation and synthesis in unirradiated cells with those which had been UV-irradiated with 500 ergs/mm*, a dose sufficient to cause 92% loss of viability. Possible effects of UV on pool sizes was checked by comparisons of the ratios of 3H and “C levels in TCA soluble material extracted from parallel cultures. However, this method does not determine alteration in levels of deoxythymidine triphosphate (d’ITP) and S-adenosylmethionine. To remedy this, different ratios of the specific activities of [methyl-3H]-L-methionine and [methyl-‘4C’J thymidine were included in the culture medium post-irradiation in experiments I, II and III. That there was no sign&ant deviation in the results of:

The DNA target for methylation It was of interest to determine whether cytosine moieties were being methylated in DNA which was synthesized after irradiation, or in the DNA that was present prior to UV-irradiation, or in both. DNA synthesized after UV irradiation was

3H dpm in 5m-Cyt from UV irradiated cell DNA “C dpm in Thy I

Sample 1 1/2Hr. Control 1 1/2Hr. UV-irradiated 3 Hr. Control 3 Hr. UV-irradiated

291

ef al.

using the different ratios of isotopes suggests that concentrations of intermediates in the synthesis of these products from methionine and thymidine were not altered by UVirradiation. It may be concluded therefore, from the correlations of these experiments, and those reported in Table 2 that these precautions were adequate to discount any alterations in pools as a result of UVirradiation. Methylation of cytosine residues in unirradiated cells was to the extent of about one methylated nucleotide per 120 DNA nucleotides. These results suggest that the extent of formation of 5-methyl cytosine relative to de novo synthesis was increased in a dose dependent manner, this being cumulatively more at 3 hr than at 1.5 hr post-irradiation, (Table 1). This effect was shown to be independent of the precursor used to measure DNA synthesis.

Relative DNA concentrations were obtained by measuring, spectrophotometrically, the UV-absorbance of aqueous solutions of the major bases scraped from the thin layer plates. Radioactivity of aqueous suspensions of the UV-absorbing areas scraped from the plates was measured by liquid scintillation counting, in Bray’s fluid.

Table 1. Synthesis and methylation

LOW

3H dpm in 5m Cyt from unirradiated ‘*C dpm in Thy

cell DNA;

of HeLa cell DNA after UV-irradiation

‘H dpm 5 methylcytosinelunit

DNA

14C dpm Thymidinelunit DNA

Expt. I

II

III

I

II

720

‘2320

2350

1980

10600

420

630

970

560

1550

5640

4320

420

1180

1640

III

% Relative extent of methylation I

II

III

7660

100

100

100

1710

1680

122

177

125

3830

19040

12950

100

100

100

510

3140

2000

143

148

195

Cells were UV-irradiated with 0 or 5OOergs/mm*, were then labelled with [methyl-3Hl-Lmethionine and [methyl-“Cl-thymidine for 1.5 or 3 hr, and DNA was extracted and purified as described in Materials and Methods. In experiments I, II and III different specific activity of ‘H-methionine relative to specific activity of “C-Thy was used. Base analysis by thin layer cellulose chromatography indicated 5-methylcytosine formation as well as thymidine incorporation. The effect of UV was measured by comparing the ratios of 5-methylcytosine to thymidine from UV-irradiated cultures to those of control cultures.

Radiation Oncology 0 Biology 0 Physics

292

Table 2. Synthesis and methylation

UV-dose ergs/mm’ 0

125 250 500

Vol. 1, No. 34

of HeLa cell DNA in response to UV-dose

‘H dpm 5 methylcytosine/unit

DNA

“C dpm Thyminelunit DNA

% Relative extent of DNA methylation

Expt. I

II

III

I

II

III

I

II

III

1340 1160 980 380

5790 3560 2860 2480

2100 1280 860 440

3510 1840 1320 480

1660 1160 720 490

1530 920 630 320

100 131 154 167

100 137 150 190

100 130 149 212

The procedure was as described for Table 1. The cultures received increasing doses of UV. Incubation time with isotopes was 3 hr.

simultaneously labelled with isotope and density-labelled with bromodeoxyuridine. Cultures were treated as follows: One was labelled for 48 hr with [2-“Cl-cytidine prior to the irradiation, thus labelling “old” DNA (Fig.

1). The second group was incubated with [2“Cl-@dine and BUdR after irradiation, to produce “C-labelled, heavy, newly synthesized, DNA. As expected there was no incorporation of “C cytidine into “old” DNA. I3

P

6n

0

‘0 - 2 X I

2

E

.-

‘I U 9

1 I

Old

lb

2b

3b

0

10

-

‘;i

20

30 TOP

BOTTOM FRACTION

NUMBER

Fig. 1. CsCl density gradient analysis of DNA from HeLa Cells, UV-irradiated with varying doses. UV-irradiation, isotope labelling, DNA preparation and CsCl analysis were as described in Materials and Methods. Samples from the gradients were alkali-treated, to completely DNA peaked at digest contaminating RNA before counting. “New, BrUdR-containing” fractions 8 and 9; “Old, light “C-Cyt-containing: DNA peaked at fractions 21-22; (a) Control, no irradiation; (b) 125 ergs/mm UV; (c) 250 ergs/mm’ UV; (d) 500 ergs/mm* UV. 0, Ezm nm; 0, ‘H dpm; A. “C dpm. Similar figuresfor UV-absorbance and’H-incorporation, but showing different extents of incorporation of “C in the BrUdR-containing DNA peaks were obtained when [2-“Cl-cytidine was present during the 4 hr incubation post UV (see Fig. 2).

Methylation of DNA in HeLa cells after ultraviolet irradiation 0 M. Low et al.

In each case, fractions containing heavy and light DNA were pooled separately, and the DNA was analyzed for base content. Recoveries of isotopes in bases, from the chromatograms, corresponded in ratio to those for DNA methylation and synthesis calculated directly from the gradients. With increasing dose of UV, DNA synthesis and the methylation of this DNA were increasingly inhibited, the latter to a greater extent than the former. After low doses of UV, the ability to methylate DNA present prior to irradiation was not, (or was only slightly) impaired. Lest these findings on DNA methylation stemmed from the presence of BrUdR, different doses of this chemical were tested for their effect on the methylation of DNA. As shown in Table 3, BrUdR caused no alteration in the ratios of nucleotide incorporation to 5methylcytosine formation. Table 3. Effect of BrUdR on DNA methylation

293

synthesis in rodent cells’.4.‘4 in Lilium,” in Physarum polycephalum 9 and in developing sea urchin.’ This phenomenon which is apparently not species specific is here shown to occur in HeLa cells also (Figs. 1 and 2). Since the cells used in these studies were unsynchronized, exponentially growing cultures, DNA synthesis, and DNA methylation would be expected to be taking place in a fraction of them at all times. Thus the continued formation of 5-methylcytosine after partial or complete inhibition of DNA synthesis by UV-irradiation (Fig. 2) is likely to be all, or in part, due to “continued” methylation. It would be of interest to determine any possible variation in DNA damage and repair, and DNA methylation, at different times during the cell cycle. Ryan and Borek have previously reported excess and altered DNA methylation in different strains of E. coli, which had received sufficient doses of UV-irradiation to inhibit

“C dpm ‘H dpm

Additions Control + IO-’ M-BrUdR + lo+’ M-BrUdR + 1O-’M-BrUdR

A& /Unit DNA 1010 850 790 660

5-m?C yt /Unit S-meCyt DNA Ade 19400 17320 16850 13800

19.1 20.3 21.3 20.6

HeLa cells were incubated for 4 hr in the presence of BrUdR, [Methyl-3H]-Methionine and [“Cl-deoxyadenosine. DNA was extracted and purified. Base analysis by thin layer cellulose chromatography indicated Smethylcytosine formation as well as adenine incorporation. The effect of BrUdR was measured by comparing the ratios of 5-methylcytosine to adenine from BrUdR-treated cultures to those of control cultures.

DISCUSSION DNA synthesis and methylation are closely related processes. Several studies on cells of rodent origin, have shown that there is a lag of about 2 min between DNA synthesis and the initiation of methylation,3.‘2 and that the extent of DNA methylation that ensues is dependent on the phase of the cell cycle, and on whether satellite or major species of DNA are being synthesized.“‘2”o Methylation of DNA can continue for several hours after its

0

100

UV-DOSE

200

A00

300

(ergs/mm

500

2)

Fig. 2. Extent of synthesis and methylation of “old” and “new” DNA in HeLa cells, in response to UV-irradiation. The procedure was as described for Fig. 1. Average measurements from 4 experiments, with standard deviations, are plotted. Measurements were related to unit UV-absorbance of DNA on the gradient, and to values from u&radiated cultures. Summation of values minus background (obtained by extrapolation of curves) was used for optical density and disintegrations per minute estimations from the gradients. These values corresponded to those obtained from chromatographic analysis of DNA from gradients. ?,?methylation of “old” DNA; 0, methylation of “new” DNA; A, synthesis of DNA.

294

Radiation Oncology 0 Biology ??Physics

DNA synthesis.18.‘9 The studies reported here indicate that the methylation of DNA is aberrant in eukaryotes exposed to irradiation, as well. What, if any, damage can originate from such aberrant methylation is obscure at present and will be clarified only when more of the functions of the methyl groups in DNA are identified. In microorganisms, some endonucleases-restriction enzymes-are known which are signalized for their nucleolytic activity by the absence of a methyl group in a certain sequence of nucleotides in DNA

Vol. 1. No. 3-4

foreign to the host. Chemically alkylated DNA has been shown to be a substrate for a nuclease from Micrococcus lysodeikticus which introduces single strand breaks into methylated DNA.= However methyl groups introduced by chemical means are preponderately on purine moieties. Whether nucleases exist in eukaryotes which recognize methylated cytosines is under study. Note added in proof. A methyl dependent DNA endonuclease has recently been demonstrated in Diplococcus pneumoniae by S. Lacks.”

REFERENCES 1. Adams, R.L.P.: The relationship between DNA 13. Lacks, S., Greenberg, B.: A deoxyribonuclease synthesis and DNA methylation in mouse of LXplococcus pneumoniae specific for methyfibroblasts. Biochim. Biophys. Acta 254: 205lated DNA. /. Biol. Chcm. 2S& 4o60-4066.1975. 212, 1971. 2. Adams, R.L.P.: Delayed methylation of DNA in developing sea urchin embryos. Nature New Biol. 244: 27-29, 1973. 3. Adams, R.L.P.: Newly synthesized DNA is not methylated. Biochim. Biophys. Acta 335: 365373, 1974. 4. Adams, R.L.P., Hogarth, C.: DNA methylation in isolated nuclei: old and new DNAs are methylated. Biochim. Biophys. Acru 331: 214220, 1973. 5. Arber, W.: Host specificity of DNA produced by Escherichia coli-V. The role of methionine in the production of host specilicity. J. Mol. Biol. 11: 247-256, 1%5. 6. Boyer, H.W., Chow, L.T., Dugaiczyk. A., Hedgpeth, J., Goodman, H.M.: DNA substrate site for the Eco restriction endonuclease and modification methylase. Nature New Biol. 244: 40-43, 1973. 7. Cleaver, J.E., Painter, R.B.: Evidence for repair replication of HeLa cell DNA damaged by ultraviolet light. Biochim. Biophys. Acta 161: 552-554, 1968. 8. Edenberg. H.E., Hanawalt, P.: Size of repair patches in the DNA of ultraviolet irradiated HeLa cells. Biochim. Biophys. Acta 272: 361-372, 1972. 9. Evans, H.H., Evans, T.E., Littman, S.: Methylation of parental and progeny DNA strands in Physarum polycephalum. I. Mol. Biol. 74: 563-572, 1973. 10. Gold, M., Hun&z, J.: The enzymatic methylation of the nucleic acids. Cold Spring Harbor Symp. Quant. Biol. 28: 149-156, 1%3. 11. Hotta, Y., Hecht, N.: Methylation of Lilium DNA during the meiotic cycle. Biochim. Biophys. Acta 238: 50-59, 1971. 12. Kappler, J.W.: The kinetics of DNA methylation in cultures of a mouse adrenal cell line. J. Cell. Physiol. 75: 21-31, 1970.

14. Lutz, D., Grahn, H.. Kroger, H.: Methylation of deoxyribonuckic acid in regenerating rat liver. 2. Naturforsch. 28: 460-462, 1973. 15. Marmur, J.: A procedure for isolation of deoxyribonucleic acid from micro-organisms. J. Mol. Biol. 3: 208-218, 1961. 16. Meselson, M., Juan, R.: DNA restriction enzyme from E. coli. Nature 217: 1110-l 114, 1968. 17. Painter, R.B., Umber, J.S., Young, B.R.: Repair replication in diploid and aneuploid human cells. Normal replication of repaired DNA after Radiat. Res. 44: ultraviolet irradiation. 133-145, 1970. 18. Ryan, A.M., Borek, E.: The enhancement of the methylation of DNA by ultraviolet irradiation. Fed. Proc. 23: 374, 1964. 19. Ryan, A.M., Borek, E.: Methylation of DNA in ultraviolet irra$iated bacteria. Biochim. Biophys. Acta 2& 203-214, 1971. 20. Schneiderman, M.H., Billen, D.: Methylation of rapidly reannealing DNA during the cell cycle of Chinese hamster cells. Biochim. Biophys. Acta 368: 352-360, 1973. 21. Smith, J.D., Arber, W., Kuhnlein, U.: Host specificity of DNA produced by Escherichia coli-XIV. The role of nucleotide methylation in in vivo. B-specific modification. J. Mol. Biol. 63: 1-8, 1972. 22. Sneider, T.W., Potter, V.R.: Methylation of mammalian DNA: Studies on Novikoff Hepatoma cells in tissue culture. J. Mol. Biol. 42: 271-284, 1%9. 23. Strauss, B.S., Robbins, M.: DNA methylated in vitro by a monofunctional alkylating agent as a substrate for a specific nuclease from Micrococcous lysodeikticus. Biochim. Biophys. Acta 161: 68-75, 1968. 24. Whitfield. B.L., Billen, I).: In vivo methylation of Escherichia coli. DNA following ultraviolet and X-irradiation. J. Mol. Biol. 63: 363-372, 1972.