Repair of single-strand breaks in normal and trisomic lymphocytes

Mutation Research, 105 (1982) 417-422

417

Elsevier BiomedicalPress

Repair of single-strand breaks in normal and trisomic lymphocytes Jay C. L e o n a r d a and T i m o t h y Merz b aThe Johns Hopkins Oncology Center, Section of Radiobiology, 600 North Wolfe Street, Baltimore, MD 21205, and bMedical College of Virginia, Virginia Commonwealth University, Department of Radiology, Division of Radiology, lOth and Marshall Streets, Richmond, VA 23298 (U.S.A.)

(Accepted 18 July 1982)

The increased incidence of acute leukemias associated with Down's syndrome has stimulated interest in the response of trisomic cells to clastogens. An increased frequency of chromosomal aberrations has been observed in peripheral blood lymphocytes from persons with Down's syndrome, trisomy 21, compared to lymphocytes from normal donors exposed in vitro to ionizing radiation (Chudina et al., 1966; Dekaban et al., 1966; Kucerova, 1967; Chudina, 1968; Sasaki and Tonumura, 1969; Sasaki et al., 1970; Lambert et al., 1976) and certain chemical clastogens (O'Brien et al., 1971), as well as after in vivo exposure to viruses (Higurashi et al., 1975, 1976). Recently, Athanasiou and colleagues (1981) reported a deficiency in the capacity of lymphocytes from persons with Down's syndrome to repair single-strand DNA breaks. They found that 1 h after exposure to 160 Gray, repair processes had restored the sedimentation profile of DNA from normal lymphocytes to control values, whereas the relative average molecular weight of DNA from irradiated lymphocytes from persons with Down's syndrome showed no increase during the repair interval. They have suggested that their data, in conjunction with the earlier data concerning the frequencies of induced chromosomal aberrations in lymphocytes from persons with Down's syndrome, reflect a decreased efficiency in some aspect of DNA repair in trisomic cells. However, for further studies of this hypothesis, it is more appropriate to study the rejoining of DNA single-strand breaks after doses comparable to those used in tests for chromosomal aberrations. Methods Repair o f D N A single-strand breaks

The repair of DNA single-strand breaks was assayed by the $1 nuclease method of Sheridan and Huang (1977), which can detect approx. 1 break per 109 dalton 0165-7992/82/0000-0000/$02.75 © Elsevier BiomedicalPress

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of single-strand DNA. This method provides a sensitive determination of singlestrand breaks induced by doses of radiation in the range used in the study of chromosome aberrations. The $1 nuclease technique is a variation of the methods of Ahnstrom and Erixxon (1973) and Rydberg (1975). Each of these methods determines the number of singlestrand breaks in DNA from the extent of strand separation in alkali. The fraction of DNA which has separated after a given interval in alkali solution is proportional to the number-average molecular weight of the DNA. Single-strand breaks reduce the effective number-average molecular weight by creating additional origins of unwinding. The theory of DNA-strand separation in alkali has been rigorously developed by Ahnstrom and Erixxon (1973) and Rydberg (1975). To quantitate the amount of unwinding, double-stranded DNA must be separated from singlestranded DNA. Sheridan and Huang's modification of the alkaline unwinding method utilizes S1 nuclease which digests single-stranded DNA. We have compared DNA single-strand break repair after exposure in vitro to 4 Gray in lymphocytes from 3 normal and 3 trisomic donors, all in their twenties and in good health. Leukocytes were isolated from buffy coat preparations. Approx. 15 × 106 leukocytes were cultured in 100 ml of McCoy's 5A + 20°70 fetal calf serum + 2070 PHA + 0.1 mCi/ml [3H]methylthymidine. The cultures were incubated for 72 h. The cells were collected by centrifugation, washed with Hank's basic salt solution, resuspended in 3H-Tdr-free media and divided into 9 centrifuge tubes at a cell concentration of 105 ceils per ml of media. One control was not exposed to radiation. All other cultures were exposed to 4 Gray of 6°Co 3'-rays. A second control was chilled prior to irradiation to prevent any repair of single-strand breaks. All other cultures were irradiated at 37°C. Approx. 1 min was required to irradiate each sample and return it to the incubator. The cultures cooled to 35°C during this period. The temperature was restored rapidly in the incubator. The cultures were allowed to repair for increasing intervals of time - 2, 5, 10, 15, 20, 30, 60 min. Repair was stopped by the addition of ice-cold Hank's basic salt solution and the tubes were placed on ice. The cells were pelleted by centrifugation in a refrigerated centrifuge, and the volume was adjusted to provide a suspension of 5 x 106 cells per ml. The unwinding assay was performed in triplicate for each repair interval. An Eppendorf pipette was used to inject 50/~1 of the cell suspension into 2.5 ml of unwinding solution (0.3 M NaOH; 0.9 M NaCI, pH 12.0), which lyses the cells, and allows dissociation of the DNA helix. We followed Sheridan and Huang's protocol (1977) regarding alkaline unwinding and $1 nuclease digestion of the crude cell lysates. The addition of ice-cold 14°70 trichloroacetic acid terminated the S~ nuclease digestion and precipitated the DNA in the samples. The samples were collected on 0.22-/z millipore filters and washed twice with 5 ml of 7070 TCA, and dried. The amount of DNA on each filter was quantitated by liquid scintillation counting. The fraction of doublestranded DNA (In) remaining was determined by the ratio of counts in the aliquot treated with S~ nuclease to the counts in the untreated aliquot. It is assumed that

419

a minimal amount of rejoining of single-strand breaks occurs in the chilled irradiated control (C0. This sample approximates the unwinding that would occur in an unrepaired sample. The unirradiated control sample ( C u ) is assumed to represent the amount of unwinding that would occur if repair were complete. The range between these is standardized to 100°70. The fraction of repair that occurred at each intervening point was calculated by the formula R n = I n - C i / C u - C u .

Results No significant differences were observed between normal and trisomy 21 lymphocytes in the amount of single-strand breaks induced by 4 Gray of C°Coirradiation or in the rate of their rejoining. When normal and trisomic lymphocytes were irradiated on ice to prevent repair, similar fractions of double-stranded DNA remained after $1 nuclease digestion, indicating that similar numbers of singlestrand breaks are induced in each (Table 1). The percentage of single-strand breaks repaired as a function of time has been plotted in Fig. 1. First-order rate constants have been calculated from the linear regression of log (1 - R n ) vs. time. The values are 0.026 min- 1 and 0.025 - 1 for normals and trisomics respectively. The Pearson product-moment correlation coefficient (0.9795) calculated from the regression of mean values for trisomics upon normal values indicates that the repair curves for normals and trisomics are the same (p<0.0001).

TABLE 1 P E R C E N T A G E OF DOUBLE-STRANDED St NUCLEASE-RESISTANT DNA R E M A I N I N G D o w n ' s syndrome

Normal

WT

VR

LH

X

Controls, no radiation

70.6

76.9

69.4

4 G at 4°C

32.5

35.5

47.3 51.4 57.6 60.3 63.3 65.2

43.2 54.6 64.2 61.2 67.3 75.2

S.E.

FB

TB

MW

X

S.E.

72.3 ± 4.0

75.0

68.1

80.4

74.5 ± 8.6

30.5

32.8 ± 2.5

30.0

34.9

40.5

35.1 ± 5.2

40.0 42.6 56.7 57.2 69.6 66.7

43.5±3.7 49.5 ± 6.2 59.5_+4.1 59.6 + 2.1 66.7 _+3.2 69.0±5.4

39.0 55.0 56.0 59.0 65.0 74.8

44.6 47.4 55.8 60.0 61.6 64.9

5.15 61.0 63.8 65.4 68.9 73.9

45.0_+6.7 54.5 ± 6.8 58.5± 1.8 61.5 ± 3.4 65.2 +_3.7 71.2_+5.5

4 G at 37°C (min of repair) 2 5 l0 15 30 60

420

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MINUTES OF" REPAIR Fig. 1. DNA strand-break rejoining. Each point represents the mean of experiments with lymphocytes from 3 different donors. Circles, trisomics. Crosses, normals.

Discussion

Our results for both normal and trisomic lymphocytes are similar to those previously reported for normal fibroblasts (Sheridan and Huang, 1977) and lymphocytes (Birnboim and Jevack, 1981). Our findings indicate that defective repair of DNA single-strand breaks and alkali-labile lesions is not the cause of the increased frequencies of ionizing radiation-induced chromosomal aberrations observed in trisomic lymphocytes. We can offer no rationale for the apparent conflict between the inability of trisomy 21 lymphocytes to repair single-strand breaks after exposure to 160 Gray dose of 3,-irradiation with the normal level of repair they exhibit after exposure to 4 Gray. However, our results are consistent with previous cytological data. A simple defect in the physical repair of DNA single-strand breaks of alkali-labile lesions cannot in itself explain the elevated levels of chromosomal aberrations induced in trisomy 21 lymphocytes by exposure to radiation. Previous studies have defined the effects of unrepaired single-strand breaks on chromosome morphology. Treatment of cells during S phase with 5-fluorodeoxyuridine produces gaps in the daughter strands of DNA. These appear as achromatic lesions (chromatid gaps) at metaphase (Taylor et al., 1962). Similarly, the fragmented appearance of prematurely condensed S phase chromosomes is also due to incomplete synthesis of daughter strands (Johnson and Rao, 1970). Chromatid and isochromatid lesions may be derived from single-strand breaks. Substitution of the DNA of V79 ceils with

421 5-bromodeoxyuridine (BrdU) during one S phase followed by photolysis during the subsequent G~ phase produced achromatic lesions, chromatid deletions, and exchanges and isochromatid deletions, whereas photolysis during G2 produced only achromatic lesions and chromatid deletions (Bender et al., 1973). Bender and his associates have hypothesized that the chromatid deletions result from cleavage of single-strand breaks by single-strand nuclease acitvity and, in the case o f the cells photolysed in Gx, the exchange aberrations result f r o m the repair of these derived double-strand breaks by recombinational or postreplicational DNA-repair mechanisms. Therefore, if single-strand break repair capacity is impaired, one would expect elevation of these lesions after irradiation of trisomy 21 lymphocytes in G1 or G2. We have recently completed such a study. We find no increase in the frequencies of achromatic lesions or chromatid deletions (neither were increases observed in chromatid exchanges or isochromatid deletions) in trisomy 21 lymo phocytes compared to normal lymphocytes after exposure to 6°Co-irradiation during G2 (Leonard and Merz, 1982). Furthermore, although we observed increased c h r o m o s o m a l radiosensitivity during G1, it is due to an increased frequency of chromosome-type aberrations as in previous studies. Increased radiosensitivity is not observed in the trisomy 21 fibroblasts, even when such diverse endpoints as the frequencies of c h r o m o s o m a l aberrations (Leonard and Merz, 1982), cell killing and mutation (Yotti et al., 1980), or the ability to rejoin D N A single-strand breaks are monitored. Increased chromosomal radiosensitivity has been successfully demonstrated in the trisomy 21 lymphocyte shortly before or after phytohemagglutinin stimulation, in vitro. These data might be explained if factors operative in the trisomy 21 lymphocyte at this time indirectly affect the amount of damage induced a n d / o r the efficiency o f repair, but the repair systems themselves are normal. Our study of the repair of single-strand breaks in the D N A of lymphocytes from persons with trisomy 21 supports this conclusion.

References Athanasiou, K., E.G. Sideris and C. Bartsocas (1980) Decreased repair of X-ray induced DNA singlestrand breaks in lymphocytes in Down's syndrome, Pediat. Res., 14, 336-338. Bender, M.A, J.S. Bedford and J.B. Mitchell (1972) Mechanisms of chromosomal aberration production, If. Aberrations induced by 5-bromodeoxyuridineand visible light, Mutation Res., 20, 403-416. Birnboim, H.C., and J.J. Jercak (1981) Fluorometric method for rapid detection of DNA strand breaks in human white cells produced by low doses of radiation, Cancer Res., 41, 1889-1892. Chudina, A.P. (1968) A study of radiosensitivity of chromosomes in normal persons and in cases of Down's disease, Genetika, 6, 99-110. Chudina, A.P., T.S. Malyutina and H.E. Pogosyane (1966) Comparative radiosensitivity of chromosomes in the cultured peripheral blood lymphocytes of normal donors and patients with Down's syndrome, Genetika, 4, 51-63. Dekaban, A.S., R. Thron and J.K. Streusing (1966) Chromosomal aberrations in irradiated blood and blood cultures of normal subjects and of selected patients with chromosomal abnormality, Radiat. Res., 27, 50-63.

422 Higurashi, M., T. Tamura and T. Nakatake (1973) Cytogenetic observations in cultured lymphocytes from patients with Down's syndrome and measles, Pediat. Res., 7, 582-587. Higurashi, M., A. Tada, S. Miyahara, M. Hirayame, H. Hoshin and T. Tamura (1976) Chromosome damage in Down's syndrome induced by chicken pox infection, Pediat. Res., 10, 189-192. Johnson, R.T., and P.N. Rao (1970) Mammalian cell fusion induction of premature chromosome condensation in interphase nuclei, Nature (London), 226, 717-722. Kucerova, M. (1967) Comparison of radiation effects in vitro upon chromosomes of human subjects, Acta Radiol., 6, 441-448. Lambert, B., K. Hansson, T.H. Bui, F. Funes-Cravioto, J. Lindsten, M. Homberg and R. Strausmanis (1976) DNA repair and frequency of X-ray and U.V. light induced chromosome aberrations in leukocytes from patients with Down's syndrome, Ann. Genet. (London), 39, 293-303. Leonard, J.C., and T. Merz (1982) Influence of cell cycle kinetics on the radiosensitivity of Down's syndrome lymphocytes, submitted. Leonard, J.C., and T. Merz (1982) Chromosomal aberrations in irradiated Down's syndrome fibroblasts, submitted. O'Brien, R.L., P. Poon, E. Kline and J.W. Parker (1971) Susceptibility of chromosomes from patients with Down's syndrome to 7,12-dimethylbenz[a]anthracene-induced aberrations in vitro, Int. J. Cancer, 8, 202-210. Rydberg, B. (1975) The rate of strand separation in alkali of DNA of irradiated mammalian ceils, Radiat. Res., 61, 274-287. Sasaki, M.S., and A. Tonomura (1969) Chromosomal radiosensitivity in Down's syndrome, Jpn. J. Hum. Genet., 14, 81-92. Sasaki, M.S., A. Tonomura and S. Matsubara (1970) Chromosome constitution and its bearing on the chromosomal radiosensitivity in man, Mutation Res., 10, 617-633. Sheridan, R.B., and P.C. Huang (1977) Single strand breakage and repair in eukaryotic DNA as assayed by $1 nuclease, Nucleic Acids Res., 4, 299-318. Taylor, J.H., W.F. Hart and J. Tung, Effects of fluorodeoxyuridine on DNA replication, chromosomal breakage and reunion, Proc. Natl. Acad. Sci. (U.S.A.), 48, 190-198. Treton, J., A. Boucays, H. Dersakissian, A. Boue and Y. Courtois (1978) The repair of DNA single strand breaks in human cells with genetical disorders, Cell Biol., Int. Rep., 2, 403-410. Yotti, L.P., W. Glover, J.E. Trosko and D.J. Segal (1980) Comparative study of X-ray and UV induced cytotoxicity, DNA repair, and mutagenesis in Down's syndrome and normal fibroblasts, Pediat. Res., 14, 88-92.