Mutation Research, 256 (1991) 295-302

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© 1991 Elsevier Science Publishers B.V. All rights reserved 0921-8734/91/$03.50

MUTAGI 00170

A re-examination of the effects of ionizing radiation on lifespan and transformation of human diploid fibroblasts Robin Holliday Genetics Division, National Institute for Medical Research, Mill Hill, London NW71AA (U.K.) and CS1RO Laboratory for Molecular Biology, Dit'ision of Biomolecular Engineering, North Ryde, NSW 2113 (Australia)

(Accepted 23 July 1991)

Keywords: Human fibroblasts; Cellular ageing; Ionizing radiation; Transformation

Summary Human diploid fibroblasts, strain MRC-5, were sequentially irradiated with 6°Co y rays at intervals during their in vitro lifespan. The results indicate that 3 or 6 doses of 1 Gy can increase lifespan, and the same was true for cells treated with 3 doses of 3 Gy. Higher doses (5 x 3 Gy) did reduce growth potential, suggesting either that mid-late passage cells become more sensitive to radiation, or that doses beyond a given threshold reduce population lifespan by multiple cellular hits. The life extension induced by 3' rays might be due to an induced hypermethylation of DNA. Alternatively, oxygen radicals produced by irradiation might trigger an adaptive stress response which would remove damaged macromolecules and thereby increase the cells' growth potential. Whichever explanation is correct, the results show that the human fibroblast system is not appropriate for the study of the well known effect of ionizing radiation in shortening the lifespan of experimental animals. Contrary to earlier published results, populations of cells treated with cumulative doses of 15 Gy or 18 Gy and held for nearly 3 months after they had reached senescence (Phase III), produced no loci of transformed cells.

It is well known that ionizing radiation significantly reduces the lifespan of mice (reviewed by Comfort, 1979). Although there there has been much debate about the phenotype of prematurely aged animals in comparison to those ageing naturally, a particularly well documented study by Lindop and Rotblatt (1961), which included postmortem examination of all the mice, strongly indicated that ionizing radiation did in some way

Correspondence: Dr. Robin Holliday, CSIRO Laboratory for Molecular Biology, Division of Biomolecular Engineering, P.O. Box 184, North Ryde, NSW 2113 (Australia).

accelerate the ageing process. Since the major known biological effect of ionizing radiation is to produce chromosome abnormalities and mutations, these longevity experiments with mice provide some support for the somatic mutation theory of ageing (for recent reviews, see Kirkwood, 1989; Rattan, 1989). Experiments have also been carried out on the effects of ionizing radiation on cultured human fibroblasts, which have finite division potential and have been widely accepted as a valid model for the experimental study of cellular ageing (Macieira-Coelho et al., 1977; Icard et al., 1979). Cultures were treated sequentially with 1 Gy (100

296 rad) of 3' radiation at intervals throughout their normal lifespan. The results were somewhat variable, but there seemed to be a significant lifeshortening with the highest doses of radiation (4-5 Gy). The effects were relatively much less than equivalent doses on the lifespan of mice. In addition to the induction of chromosome breaks and mutation, it is possible that ionizing radiation has other effects on DNA. Experimental studies on cell transformation and tumourigenesis in mice suggest that such radiation can induce heritable changes in DNA at frequencies far higher than expected from mutation (Kennedy et al., 1980; Nomura, 1982). It was later proposed that these induced changes might be due to epigenetic defects associated with alteration of the normal pattern of DNA methylation (Holliday, 1987). It is known that the overall level of D N A methylation continually declines during the serial subculture of primary diploid fibroblasts (Wilson and Jones, 1983; Fairweather et al., 1987; Matsumura et al., 1989). It is also known that a single treatment of young cells with the potent demethylating agents 5-azacytidine or 5-azadeoxycytidine strongly reduces subsequent lifespan, which means that the induced effect is heritable (Holliday, 1986a; Fairweather et al., 1987). If ionizing radiation induces significant demethylation of DNA, it would also be expected to reduce the lifespan of human diploid cells. Cultured human diploid cells very rarely, if ever, spontaneously transform to immortal permanent lines, although there is strong selection for this phenotype in senescent cultures. In an extensive series of experiments Namba et al. (1978, 1981, 1985) reported that multiple treatment of human diploid cultures with 3' rays could occasionally induce transformation and immortalization. They reported similar results with repeated treatments of 4NQO (4-nitroquinoline 1oxide), but they and others have failed to induce human cell transformation with a variety of other D N A damaging agents (see McCormick and Maher, 1988, 1989). The present experiments were undertaken to document quantitatively the effects of 6°Co y rays on the lifespan of the well characterised human foetal lung fibroblast strain MRC-5, and also to screen treated populations for loci of

transformed cells and the emergence of permanent lines. Materials and methods

Cells and growth conditions The male foetal lung fibroblast strain, MRC-5, was used throughout. These cells were obtained at passage 15 from J.P. Jacobs (National Institute of Biological Standards and Control, South Mimms). Cells were grown in Eagle's minimal essential medium (F15, Gibco Biocult) supplemented with 10% foetal calf serum, penicillin and streptomycin (100 U / m l and 0.1 m g / m l respectively). Cells were incubated at 37 ° with 5% CO 2. Young cultures were split every 3-5 days, but as they became older they were either split weekly, or the medium was changed.

Determination of lifespan Cells were grown in 25-cm 2 flasks and harvested at confluence or near confluence using trypsin-versene after rinsing with phosphatebuffered saline. Dispersed cells were counted in a Coulter Counter, and subcultured with a 1:4 and 1:8 split ratio during the period of vigorous growth, and 1:2 and 1:4 split ratio as the cells became senescent. The number of cumulative population doublings (PDs) was calculated from the known number of cells seeded (assuming 100% attachment) and the yield at each subculture. Cells reached the end of their lifespan when they failed to become confluent after 2-3 weekly changes of medium, or if the number of trypsinized cells was less than the number seeded in the flask.

Search for transformed loci Cells treated with successive high doses of radiation were transferred to 75-cm 2 flasks as they approached senescence. These cultures were kept in a confluent or sub-confluent state with weekly examination and change of medium.

Radiation treatment Cells were irradiated with a source of 6°Co 3' rays in situ 3 - 4 days after the cells were subcultured and 1-3 days before trypsinization (the exact time depending on the age of the culture),

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3xlGy~:~_~c:~ ~ ~ _ ~ : ~ ~:~ ~0 /~ j:~o~o~G ~'\\ Control~,~ ~ 6x1Gy

TABLE 1 E X P E R I M E N T A L P R O C E D U R E : DOSES O F T I O N A N D TIMES O F T R E A T M E N T

70

RADIA-

Cells were at passage 15 on day zero

y-Ray treatment

Number of cultures

None (control)

2

3 x l Gy 6x1Gy 3 x 3 Gy 5 × 3 Gy 6 × 3 Gy

:: ::

60

Times of treatment (days)

g c

50

g no further treatment ~45, 56,70 7, 25, 35 ~ n o further treatment ]53, 67 1,53, 67, 101

=_o 40

_N

30

20

at a dose rate of approximately 1 Gy (100 rad) per minute. In all cases the cells were sub-confluent. Two parallel series of cultures (A and B) were set up, and the cells received successive doses of 1 Gy or 3 Gy, with untreated controls. The radiation procedure is outlined in Table 1. Senescent cells after 5 × 3 Gy treatments were transferred to 75-cm 2 flasks and some received an additional dose of 3 Gy.

2~0

410

60

I 100

80

~ 120

I 140

I 160

I 180

Days

Fig. 1. The cumulative growth curves of an untreated population and two irradiated with 3 or 6 doses of 1 Gy. All were from experiment B (see Table 1). The arrows indicate the times of irradiation. (Note that the reduction in the growth rate of the control at the 50th PD level is in agreement with earlier results (Holliday et al., 1977, 1981).

Results The lifespans of the control and treated cultures calculated in population doublings (PDs) are listed in Table 2. The cumulative growth curves of controls and representative irradiated cultures are shown in Figs. 1 and 2. The untreated cultures had an average lifespan of 67.1 PDs which is in the expected range. Cultures treated with either 3 or 6 successive doses of 1 Gy

70

Control~. ~ ~

/ ~

A re-examination of the effects of ionizing radiation on lifespan and transformation of human diploid fibroblasts.

Human diploid fibroblasts, strain MRC-5, were sequentially irradiated with 60Co gamma rays at intervals during their in vitro lifespan. The results in...
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