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G2 chromosomal radiosensitivity in Fanconi's anemia
189
Mutation Research, 6 3 ( 1 9 7 9 ) 1 8 9 - - 1 9 9 © Elsevier/North-Holland
Biomedical Press
G2 C H R O M O S O M A L RADIOSENSITIVITY IN FANCONI'S ANEMIA *
S U S A N B. B I G E L O W
* * , J.M. R A R Y a n d M . A B E N D E R
Department of Obstetrics and Gynecology, Eastern Virginia Medical School, Norfolk, VA 23501, and Medical Department, Brookhaven National Laboratory, Upton, N Y 11973 (U.S.A.) ( R e c e i v e d 21 F e b r u a r y 1 9 7 9 ) ( R e v i s i o n r e c e i v e d 13 J u n e 1 9 7 9 ) (Accepted 18 June 1979)
Summary Both the peripheral l y m p h o c y t e s from 4 patients affected with the inherited disease Fanconi's anemia (FA), and tissue-culture fibroblasts from skin biopsies from 3 patients similarly affected were found to be a b o u t twice as sensitive to the induction of chromatid-type chromosomal aberrations by X-rays administered in the G2 phase of the cell cycle as cells from normal controls. Using tritiated thymidine labelling of peripheral lymphocytes and of cultured fibroblasts, it was determined that 3 affected patients and 3 normal controls all had similar percent labeled mitoses (PLM) curves, so the increased induced aberration yields seen in the FA cells do n o t appear to be simply a consequence of a longer than normal G2 phase of the cell cycle.
Fanconi's anemia (FA) is a rare autosomal inherited disorder, generally considered to be recessive, the principal manifestations of which include a progressive pancytopenia, malformations of the kidney, radius and thumb, and often pigmentary changes in the skin [16]. FA is also one of a small group of inherited diseases characterized b y elevated " s p o n t a n e o u s " chromosomal aberration levels [see 10,27,28]. Both FA h o m o z y g o t e s and heterozygotes are more prone to develop neoplasms, particularly leukemia, than individuals n o t carrying the gene [31]. Cells from individuals with FA and also from FA heterozygotes have * Research supported by the U.S. Atomic Energy Commission, Contract n u m b e r AT-(11-I)-2382, and be the U.S. D e p a r t m e n t of Energy. ** Material s u b m i t t e d in partial fulfillment of the requirements for the M.S. d e g r e e to the School o f Hygiene and Public Health, The J o h n s Hopkins University.
Abbreviations: AT, ataxia telangxectasia; Fa, Fanconi's anemia; PHA, phytohemagglutinin; PLM, p e r c e n t l a b e l e d mitoses.
190 been shown to be abnormally susceptible to in vitro transformation by the SV40 virus [7,17,35]. There have been a number of reports that FA cells are deficient in the ability to repair various types of DNA damage. Poon, O'Brien and Parker [20] reported that a cell line derived from a patient diagnosed as having FA was deficient in the ability to excise UV-induced pyrimidine cyclobutane dimers. Their evidence suggests a deficiency in a specific exonuclease function. However, Regan et al. (cited b y Poon, O'Brien and Parker [20]) had earlier reported that FA cells displayed a normal ability to excise dimers, so it is possible that this form of DNA-repair deficiency is not a characteristic of FA. More recently, Remsen and Cerutti [23] reported that homogenates from 2 o u t of 4 tested tissue-culture skin-fibroblast lines from FA patients were deficient in their ability to excise DNA lesions of the 5,6-dihydroxydihydrothymine t y p e from an exogenous DNA substrate. In addition, using a host-cell reactivation technique for irradiated virus, Rainbow and Howes [21] have found cells of an FA-fibroblast cell line to be deficient in the ability to repair DNA damaged by either 3,-rays or UV. Since the line was one found to be normal in the tests of Remsen and Cerutti, it may be that the defect is characteristic of all FA cells. The chromosomes of FA cells have been reported to be abnormally sensitive to clastogenic agents of several types. Schuler, Kiss and Fabian [29] found FA peripheral l y m p h o c y t e s in culture to be abnormally susceptible to chromosomal breakage by the alkylating agent tetramethansulfonil-d-manitol. Sasaki and T o n o m u r a [26] tested the sensitivity of FA lymphocytes to a variety of chemical and physical agents, including several alkylating agents, and also f o u n d a statistically significant hypersensitivity to chromosomal breakage. The most notable chromosomal susceptibility of the FA cells, however, was to the crosslinking agents nitrogen mustard, mitomycin C and 8-methoxypsoralin plus 355-nm light. Later experiments by Sasaki [25], in which effects of mitomycin C and a nonfunctional derivative, decarbamoyl mitomycin C, were compared, suggest that it is indeed the DNA crosslinks induced b y mitomycin C to which the FA cells are sensitive. A similar conclusion was reached by Fujiwara and Tatsumi [8] and Fujiwara, Tatsumi and Sasaki [9] from experiments using cell survival and DNA density-gradient centrifugation. However, Latt et al. [13] have also reported that FA cells are also abnormally sensitive to chromosomal aberration induction by a different monofunctional alkylating agent, ethylmethane sulfonate, so it appears that DNA crosslinks are n o t the only chemically-induced DNA lesions whose repair may be deficient in FA cells. Recently, Auerbach and Wolman [1 ] demonstrated that FA tissue-culture fibroblast cells are also particularly sensitive to another bifunctional agent, in this case diepoxybutane, and even more recently [2], they have reported that fibroblasts from FA heterozygotes to be more sensitive to this agent than those from normal controls. Higurachi and Conen [11,12] have reported that both peripheral lymphocytes and skin-fibroblast culture cells from individuals affected with FA display significantly higher yields of chromosome aberrations when irradiated with 3'-rays than do those from normal persons. The lymphocytes were irradiated in the Go phase of the cell cycle, prior to stimulation with phytohemagglutinin (PHA). From both the timing of the experiments and the types of aberrations
191 reported, it appears that at least the majority of the fibroblasts were in the G1 phase when irradiated. Sasaki and T o n o m u r a [26], on the other hand, were unable to demonstrate any increased susceptibility of FA peripheral l y m p h o c y t e s to chromosomal aberration production b y v-irradiation during the Go phase. Furthermore, in one experiment in which the l y m p h o c y t e cultures were irradiated 18 h prior t o harvest, in what appears from the timing and the types of aberrations observed to have been early S phase, they also failed to find any evidence of increased chromosomal radiosensitivity in the FA cells. The reports of Higurachi and Conen [11,12] and that of Sasaki and Tonomura [26] are difficult to reconcile. One difficulty with aberration-yield determinations in peripheral l y m p h o c y t e cultures, however, is that differences in cell-cycle timing can easily lead to differences in aberration frequency not necessarily directly related to intrinsic chromosomal sensitivity; the l y m p h o c y t e system in particular is b o t h complex and dynamic, and time in culture alone does not provide adequate assurance that comparable cell populations are assayed for aberrations [6]. Though aberration yields still change as a function of irradiation--fixation interval during the G2 phase of the cell cycle, the time interval involved is small, and irradiation during G2 appears to be the least complicated means of directly comparing chromosomal radiosensitivities. We therefore have carried o u t G2 irradiation experiments with both peripheral lymphocytes in culture and skin-fibroblast tissue-culture cells in an a t t e m p t to clarify the question of chromosomal radiosensitivity in FA. Materials and methods The cells used were either peripheral blood lymphocytes from FA patients, heterozygotes and normal volunteer subjects, or tissue-culture cell lines from FA patients and normal subjects. Two of the FA cell lines were obtained from the Human Genetic Mutant Cell Repository, Camden, NJ; the third was initiated from a skin biopsy from one of the patients from whom a l y m p h o c y t e sample was also obtained (FA-LE). As far as is known, none of the FA patients from w h o m l y m p h o c y t e s were obtained were related to each other. Neither are the heterozygotes the parents of any of our 4 patients. L y m p h o c y t e cultures were made by adding 0.5 ml of whole, heparinized blood to 10 ml of Eagle's minimal essential medium (Gibco) supplemented with 25% fetal calf serum (Gibco) (10% in some experiments), antibiotics and L-glutamine, to which was also added 0.25 ml of M-form PHA (Difco). Fibroblast cultures were grown in the same medium, and were subcultured using 0.25% trypsin (Gibco). Colcemid (Gibco) was added to cultures to a final concentration of 0.1 pg/ml either 2 (lymphocytes) or 3 (fibroblasts) h prior to harvest. Irradiations were done with 250 kV X-rays (h.v.1. 0.5 mm Cu) at approx. 125 R per min in air, except for the F A heterozygote experiment, for which 220 kVp X-rays (h.v.1. 2.7 mm Cu) were administered at a rate of approx. 50 R per min in air. In each experiment all treated cultures were irradiated simultaneously with the controls at r o o m temperature. Colcemid was added immediately after irradiation to all cultures, which were then reincubated at 37 ° for the 2 or 3 h prior to harvest.
192 For cultures to be used for autoradiography to obtain percent labeled mitosis (PLM) curves tritiated thymidine (New England Nuclear; 6.7 Ci/mM) was added to a final concentration of 1/~Ci per ml of medium and allowed to remain until the cultures were terminated. In the case of lymphocytes, cultures were fixed every hour following addition of the tritiated thymidine up to the 9th hour; fibroblast cultures were fixed at 1.5 h, and then each hour up to 9.5h. The cells were fixed in 3 : 1 methyl alcohol : glacial acetic acid following a 15-min hypotonic treatment with 0.075 M KC1. After washing with fresh fixative, cells were mounted by dropping the suspension into the surface of wet glass slides and then flaming. Slides for autoradiography were dipped in NTB-2 nuclear-track emulsion (Kodak) diluted 1 : 1 with distilled water, dried, exposed for one week and then developed in D-19 developer for 4 min, fixed and allowed to air dry. Slides were stained with 10% Giemsa (Harleco). Aberrations were scored at 800×, following commonly accepted criteria for the various aberration types. Metaphases were scored as labeled in the autoradiographs if there were more than 10 grains over them after subtracting an average background value determined separately for each slide by counting grains over areas not containing any cells. Results A dose--effect curve was obtained for one FA patient's lymphocytes in order to determine a single appropriate X-ray dose for later experiments. With the I 3.25
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Fig. 1. T o t a l c h r o m o s o m a l a b e r r a t i o n y i e l d s in peripheral l y m p h o c y t e s f r o m a F A p a t i e n t ( o p e n circles) and f r o m a n o r m a l c o n t r o l ( h a l f - c l o s e d circles) given various d o s e s o f X-rays in t h e G 2 phase o f the cell c y c l e . T h e single c l o s e d circle r e p r e s e n t s t h e average u m r r i d i a t e d c o n t r o l value for 7 9 2 cells f r o m 4 F A p a t i e n t s (see t e x t ) . Bars i n d i c a t e P o l s s o n errors. 1 0 0 cells w e r e s c o r e d per p o i n t for all o t h e r s a m p l e s .
193
exception of the unirradiated FA culture, 100 cells were scored per point. The results are summarized in Fig. 1. Because the unirradiated F A culture in this experiment failed to yield but a few scoreable metaphases, the point shown is the average value for all of our unirradiated FA lymphocyte cultures. The bars indicate Poisson errors. The lines shown are least-squares linear regressions. On the basis of this preliminary experiment, a "standard dose" of 50 R was adopted for 5 additional experiments with lymphocytes from 3 other patients. The results, including the control and 50-R data from the experiment shown in Fig. 1, are given in Table 1. It will be seen that in spite of some variability in magnitude, in each experiment the yields of both achromatic lesions and chromatid deletions in the F A cells are higher than in the controls. Table 2 gives a summary of the pooled data from all of the lymphocyte experiments. Because of the different sample sizes in two of the experiments, the net increases were calculated by subtracting the number of aberrations expected in a sample the size of the irradiated one on the basis of their frequency in the unirradiated one. The errors were based on Poisson variance. The differences
TABLE
1
CHROMOSOMAL LYMPHOCYTES
ABERRATIONS FROM FANCONI'S
IN IRRADIATED AND UNIRRADIATED PERIPERAL BLOOD ANEMIA PATIENTS AND NORMAL CONTROL INDIVIDUALS
Subject
Irradiation (R)
Number of cells scored
Achromatic lesions
1
FA - LE
None 50
11 100
26 189
2 47
2 36
C - 1
None 50
100 100
6 64
1 11
1 0
FA - KE
None 50
200 200
46 318
22 145
5 89
C - 2
None 50
200 200
29 93
14 28
1 12
FA - KE
None 50
150 150
41 228
14 104
5 4
C - 3
None 50
150 150
11 49
3 18
0 10
FA - BA
None 50
100 100
27 167
13 84
1 8
C - 4
None 50
100 100
11 64
9 37
1 0
FA - LA
None 50
150 150
37 272
10 106
0 10
C - 5
None 50
150 150
18 108
17 43
0 4
FA - KE
None 50
181 231
51 901
4 592
1 31
C - 4
None 50
100 285
14 775
0 437
0 44
2
3
4
5
6
a Includes isochromatid tions were seen.
deletions
and chromatid
interchanges;
Chromatid deletions
Others a
Expt.
no unequivocal
chromosome-type
aberra-
194
TABLE 2 POOLED CHROMOSOMAL ABERRATION YIELDS FROM THE 6 LYMPHOCYTE TION EXPERIMENTS FOR WHICH THE RAW DATA ARE GIVEN IN TABLE 1
G 2 X-IRRADIA-
N e t i n c r e a s e s c a l c u l a t e d b y s u b t r a c t i n g t h e n u m b e r o f e a c h t y p e o f a b e r r a t i o n e x p e c t e d in a s a m p l e t h e size o f t h e i r r a d i a t e d o n e o n the basis o f their f r e q u e n c y in t h e u n i r r a d i a t e d s a m p l e . Number o f cells
Achromahc lesions
Chromatid deletions
Other
Total
FA (4 s u b -
Unirradiated
792
228 2 8 . 8 +- 1.9 %
65 8.2 ± 1 . 0 %
14 1.8 ± 0 . 5 %
307 3 8 . 8 -+ 2 . 2 %
jeers, 6 Expts.)
Irradiated
931
2075 2 2 2 . 9 ± 4.9 %
1078 1 1 5 . 8 +- 3.5 %
178 19.1 + 1 . 4 %
3331 357.8 ± 6.2 %
1807 1 9 4 . 1 + 4.6 %
1002 1 0 7 . 6 ± 3.4 %
162 17.4 + 1.4 %
2971 3 1 9 . 1 + 5.9 %
Net increase Controls (5 s u b -
Um~adiated
800
89 1 1 . 1 + 1.2 %
44 5.5 + 0.8 %
3 0.4 ± 0.2 %
136 1 7 . 0 + 1.5 %
jects, 6 Expts.)
Irtadiated
985
1153 1 1 7 . 1 -+ 3 . 5 %
574 5 8 . 3 -+ 2.4 %
70 7.1 + 0.9 %
1797 1 8 2 . 4 + 4.3 %
1043 1 0 5 . 9 ± 3.3
520 5 2 . 8 ± 2.3 %
66 6.7 + 0 . 8 %
1629 1 6 5 . 4 -+ 4 . 1 %
Net increase
between the irradiated FA and the irradiated control cells are, of course, statistically highly significant. The raw results of our experiments with the 3 FA-fibroblast lines are given in Table 3, and summarized in Table 4. The net increases were calculated as for Table 2. While the aberration yields are lower than in the lymphocyte experiments, very likely because of a longer G2 phase, it can be seen that the fibroblast results correspond qualitatively to those for lymphocytes, and that FA fibroblasts are also characterized by a significantly elevated susceptibility to TABLE 3 CHROMOSOMAL ABERRATIONS IN IRRADIATED AND UNIRRADIATED SKIN-BIOPSY FIBROBLAST CULTURE CELLS FROM 3 FA PATIENTS AND 3 NORMAL CONTROL INDIVIDUALS Expt.
Culture
Irradiation (R)
Number of cells ~ o r e d
Achromatic lesions
Chrom~id dele~ons
Others a
1
FA - LE
0 50
100 100
13 146
7 48
3 10
C-6
0 50
100 100
13 50
9 14
3 0
GM-368
0 50
75 75
16 133
14 49
1 3
C-I0
0 50
75 75
14 24
8 15
0 0
GM-391
0 50
75 75
10 131
8 49
0 4
C-323
0 50
75 75
10 13
4 12
0 0
2
3
a Includes isochromatid deletions and chromatid interchanges; no unequivocal chromosome-type aberrations were seen.
195
TABLE 4 POOLED CHROMOSOMAL ABERRATION YIELDS FROM THE 3 FIBROBLAST G2 X-IRRADIATION E X P E R I M E N T S F O R W H I C H T H E R A W D A T A A R E G I V E N IN T A B L E 3
Number o f cells FA (3 E x p t s . )
Unirradiated
250
I*Tadiated
250
Achromatic lesions
Other
39 1 5 . 6 ± 2.5 %
4 1.6 -+ 0.8 %
410 164.0 +- 8 . 1 %
146 58.4 ± 4.8 %
17 6.8 ± 1.6 %
573 229.2 + 9.6 %
371 1 4 8 . 4 + 7.7 %
107 4 2 . 8 ± 4.1
13 5.2 ± 1.4 %
491 1 9 6 . 4 ± 8.9 %
3 1.2 ± 0.7 %
61 24.4 + 3 . 1 %
39 1 5 . 6 -+ 2.5 %
Net increase Control (3 E x p t s . )
Chromatid deletions
Unirtadiated
250
37 1 4 . 8 ± 2.4 %
21 8.4 ± 1.8 %
Irradiated
250
87 3 4 . 8 + 3.7 %
41 1 6 . 4 ± 2.6 %
50 2 0 . 0 ± 2.8 %
20 8.0 ± 1.8 %
Net increase
Total
0 --
82 3 2 . 8 + 3.6 %
128 51.2 ± 4.5 % 73 2 9 . 2 + 3.4 %
chromosomal breakage by X-rays. Because the yields of both achromatic lesions and chromatid deletions tend to decrease with increasing irradiation-fixation interval following G2 irradiation [30], the observed apparent G2 chromosomal radiosensitivity might result if the FA cells simply take longer to move from the S phase into mitosis. Indeed, such an explanation is suggested by the report of Loughman [15] of difficulty in determining cell-cycle time for lymphocytes of a FA patient because a substantial fraction of the cells would not label with tritiated thymidine, shifting the PLM curve relative to that normal controls in a manner suggestive of a lengthened G2 phase. Fig. 2 shows the PLM curves we obtained in our autoradiographic experiments both with lymphocytes from 2 FA patients and 2 normal
I I I I I CONTROLS (2) LYMPHOCYTES ~, FA (2) LYMPHOCYTES • CONTROL FIBROBLASTS • F.& F I B R O B L A S T S
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Fig. 2. P e r c e n t l a b e l e d m i t o s i s c u r v e s f o r p e r i p h e r a l l y m p h o c y t e s ( o p e n s y m b o l s ) a n d fibroblast-cell lines (closed symbols) f r o m FA patients and normal control indiwduals.
196 controls and with a single FA and a control fibroblast-cell line. It may be seen that while the average duration of G2 for the fibroblasts is longer {about 7 h) than that of the lymphocytes (about 5 h), there is little evidence that the PLM curves of FA cells are different from those of normal control cells. Certainly any difference there might be is much too small to account for differences in aberration yield of the magnitude we have observed.
Discussion Our results confirm in one sense the chromosomal sensitivity of Fanconi's anemia cells to ionizing radiation originally reported by Higurachi and Conen [11,12] based upon experiments in which peripheral lymphocytes were irradiated in the Go phase of the cell cycle. Our experiments, however, included only G2 irradiations, and thus do not resolve the conflict between their observation and that of Sasaki and Tonomura [26], who failed to observe substantially increased aberration yields in lymphocytes treated in either the Go or (probably) the S phase. Recently we have made a similar study of lymphocytes from patients with ataxia telangiectasia (AT) treated with X-rays in either Go or G2 [6]. Though we found a G2 chromosomal rasiosensitivity very similar to our present result for FA cells, we were unable to confirm the Go radiosensitivity reported by Higurachi and Conen [ 11,12] for lymphocytes from patients with this desease. It seems possible, then, in agreement with the findings of Sasaki and Tonomura, that FA cells are actually only particularly radiosensitive during the G2 phase of the cell cycle. Whatever the final resolution of the question of chromosomal radiosensitivity during the rest of the cell cycle, our G2 results seem unequivocal. That the phenomenon is a general characteristic of FA is suggested by our similar findings for both lymphocytes and fibroblasts, and for cells from 6 apparently unrelated persons with the disease. Our autoradiographic experiments appear to rule out the possibility that increased aberration yields results simply from a longer G2 phase characterizing FA cells than cells from normal individuals, and suggest that the labeling curve anomaly reported by Loughman [15] for one FA patient is not in fact a general characteristic of FA cells. Unfortunately, the present experiments cannot rule out the possibility that ionizing radiation might simply induce an abnormal delay in the progression of FA cells through G2, thus resulting in the sampling of normal and FA-cell populations irradiated at different stages within the G2 phase at any particular fixation time, and explaining the greater aberration yields in the FA cells. Nevertheless, though additional experiments are clearly needed to settle this point, this possibility seems unlikely to us. Even at AT cells, in which ionizing radiation administered during the G1 (Go) phase of the cell cycle causes markedly longer cell-cycle delays than in normal cells, exhibit little if any delay in their progression to mitosis if irradiated in the G2 [6], though AT cells do exhibit the same G2 chromosomal radiosensitivity phenomenon as FA cells [6,22,34]. The definite increases in chromosomal aberration yields induced in FA cells by G2 X-irradiation are of particular interest with respect to DNA-repair mechanisms and the molecular mechanisms involved in the induction of aberrations by ionizing radiation. If, as reported by Remsen and Cerutti [23] and by Rain-
197
b o w and Howes [21] FA cells are deficient in their ability to repair a form of DNA-base damage induced b y ionizing radiation, then the excess aberrations might be attributed to unrepaired (or partially repaired) lesions of this type, rather than, perhaps, to higher initial amounts of DNA damage. Unfortunately, however, defects in repair of other DNA lesions, including double and single polynucleotide strand breaks, cannot be ruled o u t on the basis of information presently available. Furthermore, the specific deficiency noted by Remsen and Cerutti, involving the cells' ability to excise products of the 5,6-dilhydroxyd i h y d r o t h y m i n e type, was found in only 2 of 4 FA-cell lines tested, and so may n o t be generally characteristic of the disease. The aberrations seen in both the FA and the normal cells in our experiments were all of the chromatid type, and consisted mainly of achromatic lesions and simple chromatid deletions. Only a relatively few isochromatid deletions or chromatid exchanges were seen, as is the normal consequence of irradiation of cells in G2 [30]. Thus the increased yields in the FA cells were largely, at least, in the classes both induced in normal cells by ionizing radiation and arising spontaneously in untreated cells. Several lines of evidence suggest that single polynucleotide strand breaks may appear as achromatic lesions at metaphase [3,32] and double polynucleotide strand breaks must surely at least be involved in chromatid breakage [4,18]. It furthermore seems clear that some single polynucleotide strand breaks must become double-strand breaks and contribute to chromatid breakage [3,5,18]. Thus the observed increases in achromatic lesions and in chromatid deletions in our irradiated FA cells appear consistent with the possibility that FA cells are simply deficient in their ability to repair single polynucleotide strand breaks. Nevertheless, because excision-type repair of damaged DNA bases appears to involve a number of enzymatic steps, and because a discontinuity in the polynucleotide strand under repair must exist during part of the excision process, it is also possible to reconcile the present cytogenetic results equally well with the idea that FA cells are characterized b y a defect in excision repair of damaged bases like that found by Remsen and Cerutti [23]. It would only be required that the defect involve a step after the " n i c k " is made, which could result in a larger number of single-strand breaks being present in FA than in normal cells, and thus, if achromatic lesions and chromatid deletions are in fact attributable to such breaks, in larger yields of these aberration types. The observations both of Remsen and Cerutti and of Rainbow and Howes [21] are consistent with such an idea, since the former measured the appearance of the damaged base in the acid-soluble fraction following treatment of irradiated DNA with cell or nuclear sonicates, while the latter measured host-cell reactivation of irradiated virus. The recent finding of Natarajan and Obe [18] that treatment of Chinese hamster tissue-culture cells irradiated in G2 with a Neurospora endonuclease specific for cleaving single-stranded DNA gave increased yields of chromatidt y p e aberrations observed at metaphase is also consistent, though it also suggests the alternative possibility that FA cells might simply possess more of a similar enzymatic activity than normal cells. Of course it is possible as well that a defect in a step c o m m o n to both damaged-base excision and strand-break repair could also account for all of the observations. It is of interest to compare the results for FA cells with the information
198 available on AT cells, which are in some respects similar in their response to ionizing radiation. Both FA and AT lymphocytes have been reported to be chromosomally radiosensitive to ionizing radiation during the Go phase [11,12] though as already noted we have been unable to confirm this observation for AT lymphocytes [6]. Nevertheless, precisely the same sort of G2 chromosomal radiosensitivity has been found in AT cells irradiated in G2 as we here report for FA cells [ 6 , 2 2 , 3 4 ] . Furthermore, Taylor et al. [34] have demonstrated that Go irradiation of AT lymphocytes results in a small but statistically significant yield of aberration of the chromatid type, an unexpected consequence of Go or G1 irradiations, but one which we have been able to confirm [6]. As with FA cells, a defect in the ability of AT cells to excise DNA-base damage has been reported [19], though the base damage is not of the 5,6-dihydroxydihydrothymine type deficient in at least some FA cells [24]. In addition, it has been reported that AT cells will show a normal ability to repair both single and double polynucleotide strand breaks [ 1 4 , 3 3 , 3 6 ] . It is therefore possible to conclude that the increased G2 aberration yields in irradiated AT cells arise from the base-damage repair defect rather than from a parallel polynucleotidestrand break-repair deficiency, and thus infer that this is at least possible in FA cells as well, even though the type of base damage for which excision appears to be deficient in the t w o diseases is not the same. References 1 Auerbach, A.D., and S.R. Wolman, Susceptiblhty of Fanconi's anaemia fibroblasts to chromosome damage by carcinogens, Nature (London), 261 (1976) 494--496. 2 A u e r b a c h , A . D . , a n d S.R. W o l m a n , C a r c i n o g e n - i n d u c e d c h r o m o s o m e b r e a k a g e m F a n c o n i ' s a n a e m i a h e t e r o z y g o u s cells, N a t u r e ( L o n d o n ) 2 7 1 ( 1 9 7 8 ) 6 9 - - 7 1 . 3 B e n d e r , M . A , J . S . B e d o r d a n d J . B . M i t c h e l l , M e c h a m s m s o f c h r o m o s o m a l a b e r r a t i o n p r o d u c t i o n , II. A b e r r a t i o n s i n d u c e d b y 5 - b r o m o d e o x y u r i d i n e a n d visible l i g h t , M u t a t i o n R e s . , 2 0 ( 1 9 7 3 ) 4 0 3 - - 4 1 6 . 4 B e n d e r , M . A , H . G . G r l g g s a n d J . S . B e d f o r d , M e c h a n i s m s o f c h r o m o s o m a l a b e r r a t i o n p r o d u c t i o n , III. Chemicals and ionizing radiation, Mutation Res., 23 (1974) 197--212. 5 B e n d e r , M . A , H . G . G r i g g s a n d P . L . W a l k e r , M e c h a n i s m s o f c h r o m o s o m a l a b e r r a t i o n p r o d u c t i o n , I. A b e r r a t i o n i n d u c t i o n b y u l t r a v i o l e t light, M u t a t i o n R e s . , 2 0 ( 1 9 7 3 ) 3 8 7 - - - 4 0 2 . 6 B e n d e r , M . A , J . M . R a r y a n d R . P . K a l e , M e c h a n i s m s o f c b - r o m o s o m a l a b e r r a t i o n p r o d u c t i o n IV. 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199
1 7 Miller, R . W . , a n d G . J . T o d a r o , Viral t r a n s f o r m a t i o n o f c e n s f r o m p e r s o n s a t h i g h r i s k o f c a n c e r , Lancet, i (1969) 81--82. 1 8 N a t a r a j a n , A . T . , a n d G. O b e , M o l e c u l a r m e c h a n i s m s r e v o l v e d in t h e p r o d u c t i o n o f c h r o m o s o m a l a b e r r a t i o n s I. U t i l i z a t i o n o f N e u r o s p o r a e n d o n u c l e a s e f o r t h e s t u d y o f a b e r r a t i o n p r o d u c t i o n in G 2 s t a g e o f t h e ceil c y c l e , M u t a t i o n Res., 5 2 ( 1 9 7 8 ) 1 3 7 - - 1 4 9 . 1 9 P a t e r s o n , M.C., B.P. S m i t h , P . H . M . L o h m a n , A . K . A n d e r s o n a n d L. F 1 s h m a n , D e f e c t i v e e x c i s i o n r e p a i r o f X - r a y - d a m a g e d D N A in h u m a n ( a t a x i a t e l a n g i e c t a s i a ) f i b r o b l a s t s , N a t u r e ( L o n d o n ) , 2 6 0 ( 1 9 7 6 ) 444--447. 20 Pooh, P.K., R.L. O'Brien and J.W. Parker, Defective DNA repalr in Fanconi's anemia, Nature (London), 250 (1974) 223--225. 21 R a i n b o w , A . J . , a n d M. H o w e s , D e f e c t i v e r e p a i r o f u l t r a v i o l e t a n d g a m m a - r a y - d a m a g e d D N A m F a n c o n i ' s a n e m i a , I n t . J. R a d i a t . Biol., 3 1 ( 1 9 7 7 ) 1 9 1 - - 1 9 5 . 2 2 R a r y , J . M . , M . A B e n d e r a n d T . E . K e i l y , C y t o g e n e t l c s t u d i e s o f a t a x l a t e l a n g i e c t a s i a , A m . J. H u m a n Genet., 28 (1974) 70a. 23 Remsen, J.F., and P.A. Cerutti, Deficiency of gamma-ray excision repalr in skin fibroblasts from p a t i e n t s w i t h F a n c o n l ' s a n e m i a , P r o c . N a t l . A c a d . Sci. ( U . S . A . ) , 73 ( 1 9 7 6 ) 2 4 1 9 - - 2 4 2 3 . 24 Remsen, J.F., and P.A. Ceruttl, Excision of gamma-ray induced thymine leslons by preparations from ataxia telangiectasia fibroblasts, Mutatlon Res., 43 (1977) 139--146. 2 5 Sasaki, M.S., Is F a n c o n i ' s a n e m i a d e f e c t i v e in a p r o c e s s e s s e n t i a l t o t h e r e p a i r o f D N A c r o s s links?, Nature (London), 257 (1975) 501--503. 26 S a s a k i , M.S., a n d A. T o n o m u r a , A h i g h s u s c e p t i b i l i t y o f F a n c o n i ' s a n e m i a t o c h r o m o s o m e b r e a k a g e by DNA cross-hnking agents, Cancer Res., 33 (1973) 1829--1836. 2 7 S c h m i d t , W., F a m i l i a l c o n s t i t u t i o n a l p a n m y e l o c y t o p a t h y , F a n c o n i ' s a n e m l a If. A d l s c u s s i o n o f t h e cytogenetic finchngs m Fanconi's anemia, Seminars Hematol., 4 (1967) 241--249. 2 8 S c h r o e d e r , T . M . , a n d R . K u r t h , S p o n t a n e o u s c h r o m o s o m e b r e a k a g e a n d h i g h i n c i d e n c e o f l e u k e m m in m h e r l t e d dlsease, B l o o d , 3 7 ( 1 9 7 1 ) 9 6 - - 1 1 2 . 2 9 S c h u l e r , D., A. Kiss a n d F. F a i n a n , C h r o m o s o m a l p e c u l i a r i t i e s a n d " i n v i t r o " e x a m m a t l o n s in F a n coni's anemia, Humangenetik, 7 (1969) 314--322. 3 0 S c o t t , D., a n d H . J . E v a n s , X - r a y - i n d u c e d c h r o m o s o m a l a b e r r a t i o n s in Vlcla faba: c h a n g e s in r e s p o n s e d u r i n g t h e cell c y c l e , M u t a t i o n R e s . , 4 ( 1 9 6 7 ) 5 7 9 - - 5 9 9 . 3 1 S w i f t , M . R . , F a n c o m ' s a n e m i a in t h e g e n e t i c s o f n e o p l a s i a , N a t u r e ( L o n d o n ) , 2 3 0 ( 1 9 7 1 ) 3 7 0 - - 3 7 3 . 3 2 T a y l o r , J . H . , W . F . H a u t a n d J. T u n g , E f f e c t s o f f l u o r o d e o x y u n d i n e o n D N A r e p l i c a t i o n , c h r o m o s o m e b r e a k a g e a n d r e u m o n , P r o c . N a t l . A c a d . Scl. ( U . S . A . ) , 4 8 ( 1 9 6 2 ) 1 9 { ) - - 1 9 8 . 3 3 T a y l o r , A . M . R . , D . G . H a r n d e n , C.F. A r l e t t , S.A. H a r c o u r t , S. S t e v e n s a n d B.A. Bridges, A t a x m t e l a n giectasia: a human mutation with abnormal radiation sensitivity, Nature (London), 258 (1975) 427-429. 3 4 T a y l o r , A . M . R . , J . A . M c t c a i f e , J . M . O x f o r d a n d D . G . H a r n d e n , Is c h r o m a t l d - t y p e d a m a g e in a t a x i a telangieetasia after irrachation at G O a consequence of defective repair?, Nature (London), 260 (1976) 441--443. 3 5 T o d a r o , G . J . , H. G r e e n a n d M . R . S w i f t , S u s c e p t i b i l i t y o f h u m a n d i p l o i d f i b r o b l a s t s t r a i n s t o t r a n s f o r m a t i o n o f S V - 4 0 virus, S c i e n c e , 1 5 3 ( 1 9 6 6 ) 1 2 5 2 - - 1 2 5 4 . 3 6 V i n c e n t J r . , R . A . , R . B . S h e r i d a n III a n d P.C. H u a n g , D N A s t r a n d b r e a k a g e r e p a i r m a t a x i a t e l a n g l e c t a s i a f i b r o b l a s t - l i k e cells, M u t a t i o n R e s . , 3 3 ( 1 9 7 5 ) 3 5 7 - - 3 6 6 .
Mutation Research, 6 3 ( 1 9 7 9 ) 1 8 9 - - 1 9 9 © Elsevier/North-Holland
Biomedical Press
G2 C H R O M O S O M A L RADIOSENSITIVITY IN FANCONI'S ANEMIA *
S U S A N B. B I G E L O W
* * , J.M. R A R Y a n d M . A B E N D E R
Department of Obstetrics and Gynecology, Eastern Virginia Medical School, Norfolk, VA 23501, and Medical Department, Brookhaven National Laboratory, Upton, N Y 11973 (U.S.A.) ( R e c e i v e d 21 F e b r u a r y 1 9 7 9 ) ( R e v i s i o n r e c e i v e d 13 J u n e 1 9 7 9 ) (Accepted 18 June 1979)
Summary Both the peripheral l y m p h o c y t e s from 4 patients affected with the inherited disease Fanconi's anemia (FA), and tissue-culture fibroblasts from skin biopsies from 3 patients similarly affected were found to be a b o u t twice as sensitive to the induction of chromatid-type chromosomal aberrations by X-rays administered in the G2 phase of the cell cycle as cells from normal controls. Using tritiated thymidine labelling of peripheral lymphocytes and of cultured fibroblasts, it was determined that 3 affected patients and 3 normal controls all had similar percent labeled mitoses (PLM) curves, so the increased induced aberration yields seen in the FA cells do n o t appear to be simply a consequence of a longer than normal G2 phase of the cell cycle.
Fanconi's anemia (FA) is a rare autosomal inherited disorder, generally considered to be recessive, the principal manifestations of which include a progressive pancytopenia, malformations of the kidney, radius and thumb, and often pigmentary changes in the skin [16]. FA is also one of a small group of inherited diseases characterized b y elevated " s p o n t a n e o u s " chromosomal aberration levels [see 10,27,28]. Both FA h o m o z y g o t e s and heterozygotes are more prone to develop neoplasms, particularly leukemia, than individuals n o t carrying the gene [31]. Cells from individuals with FA and also from FA heterozygotes have * Research supported by the U.S. Atomic Energy Commission, Contract n u m b e r AT-(11-I)-2382, and be the U.S. D e p a r t m e n t of Energy. ** Material s u b m i t t e d in partial fulfillment of the requirements for the M.S. d e g r e e to the School o f Hygiene and Public Health, The J o h n s Hopkins University.
Abbreviations: AT, ataxia telangxectasia; Fa, Fanconi's anemia; PHA, phytohemagglutinin; PLM, p e r c e n t l a b e l e d mitoses.
190 been shown to be abnormally susceptible to in vitro transformation by the SV40 virus [7,17,35]. There have been a number of reports that FA cells are deficient in the ability to repair various types of DNA damage. Poon, O'Brien and Parker [20] reported that a cell line derived from a patient diagnosed as having FA was deficient in the ability to excise UV-induced pyrimidine cyclobutane dimers. Their evidence suggests a deficiency in a specific exonuclease function. However, Regan et al. (cited b y Poon, O'Brien and Parker [20]) had earlier reported that FA cells displayed a normal ability to excise dimers, so it is possible that this form of DNA-repair deficiency is not a characteristic of FA. More recently, Remsen and Cerutti [23] reported that homogenates from 2 o u t of 4 tested tissue-culture skin-fibroblast lines from FA patients were deficient in their ability to excise DNA lesions of the 5,6-dihydroxydihydrothymine t y p e from an exogenous DNA substrate. In addition, using a host-cell reactivation technique for irradiated virus, Rainbow and Howes [21] have found cells of an FA-fibroblast cell line to be deficient in the ability to repair DNA damaged by either 3,-rays or UV. Since the line was one found to be normal in the tests of Remsen and Cerutti, it may be that the defect is characteristic of all FA cells. The chromosomes of FA cells have been reported to be abnormally sensitive to clastogenic agents of several types. Schuler, Kiss and Fabian [29] found FA peripheral l y m p h o c y t e s in culture to be abnormally susceptible to chromosomal breakage by the alkylating agent tetramethansulfonil-d-manitol. Sasaki and T o n o m u r a [26] tested the sensitivity of FA lymphocytes to a variety of chemical and physical agents, including several alkylating agents, and also f o u n d a statistically significant hypersensitivity to chromosomal breakage. The most notable chromosomal susceptibility of the FA cells, however, was to the crosslinking agents nitrogen mustard, mitomycin C and 8-methoxypsoralin plus 355-nm light. Later experiments by Sasaki [25], in which effects of mitomycin C and a nonfunctional derivative, decarbamoyl mitomycin C, were compared, suggest that it is indeed the DNA crosslinks induced b y mitomycin C to which the FA cells are sensitive. A similar conclusion was reached by Fujiwara and Tatsumi [8] and Fujiwara, Tatsumi and Sasaki [9] from experiments using cell survival and DNA density-gradient centrifugation. However, Latt et al. [13] have also reported that FA cells are also abnormally sensitive to chromosomal aberration induction by a different monofunctional alkylating agent, ethylmethane sulfonate, so it appears that DNA crosslinks are n o t the only chemically-induced DNA lesions whose repair may be deficient in FA cells. Recently, Auerbach and Wolman [1 ] demonstrated that FA tissue-culture fibroblast cells are also particularly sensitive to another bifunctional agent, in this case diepoxybutane, and even more recently [2], they have reported that fibroblasts from FA heterozygotes to be more sensitive to this agent than those from normal controls. Higurachi and Conen [11,12] have reported that both peripheral lymphocytes and skin-fibroblast culture cells from individuals affected with FA display significantly higher yields of chromosome aberrations when irradiated with 3'-rays than do those from normal persons. The lymphocytes were irradiated in the Go phase of the cell cycle, prior to stimulation with phytohemagglutinin (PHA). From both the timing of the experiments and the types of aberrations
191 reported, it appears that at least the majority of the fibroblasts were in the G1 phase when irradiated. Sasaki and T o n o m u r a [26], on the other hand, were unable to demonstrate any increased susceptibility of FA peripheral l y m p h o c y t e s to chromosomal aberration production b y v-irradiation during the Go phase. Furthermore, in one experiment in which the l y m p h o c y t e cultures were irradiated 18 h prior t o harvest, in what appears from the timing and the types of aberrations observed to have been early S phase, they also failed to find any evidence of increased chromosomal radiosensitivity in the FA cells. The reports of Higurachi and Conen [11,12] and that of Sasaki and Tonomura [26] are difficult to reconcile. One difficulty with aberration-yield determinations in peripheral l y m p h o c y t e cultures, however, is that differences in cell-cycle timing can easily lead to differences in aberration frequency not necessarily directly related to intrinsic chromosomal sensitivity; the l y m p h o c y t e system in particular is b o t h complex and dynamic, and time in culture alone does not provide adequate assurance that comparable cell populations are assayed for aberrations [6]. Though aberration yields still change as a function of irradiation--fixation interval during the G2 phase of the cell cycle, the time interval involved is small, and irradiation during G2 appears to be the least complicated means of directly comparing chromosomal radiosensitivities. We therefore have carried o u t G2 irradiation experiments with both peripheral lymphocytes in culture and skin-fibroblast tissue-culture cells in an a t t e m p t to clarify the question of chromosomal radiosensitivity in FA. Materials and methods The cells used were either peripheral blood lymphocytes from FA patients, heterozygotes and normal volunteer subjects, or tissue-culture cell lines from FA patients and normal subjects. Two of the FA cell lines were obtained from the Human Genetic Mutant Cell Repository, Camden, NJ; the third was initiated from a skin biopsy from one of the patients from whom a l y m p h o c y t e sample was also obtained (FA-LE). As far as is known, none of the FA patients from w h o m l y m p h o c y t e s were obtained were related to each other. Neither are the heterozygotes the parents of any of our 4 patients. L y m p h o c y t e cultures were made by adding 0.5 ml of whole, heparinized blood to 10 ml of Eagle's minimal essential medium (Gibco) supplemented with 25% fetal calf serum (Gibco) (10% in some experiments), antibiotics and L-glutamine, to which was also added 0.25 ml of M-form PHA (Difco). Fibroblast cultures were grown in the same medium, and were subcultured using 0.25% trypsin (Gibco). Colcemid (Gibco) was added to cultures to a final concentration of 0.1 pg/ml either 2 (lymphocytes) or 3 (fibroblasts) h prior to harvest. Irradiations were done with 250 kV X-rays (h.v.1. 0.5 mm Cu) at approx. 125 R per min in air, except for the F A heterozygote experiment, for which 220 kVp X-rays (h.v.1. 2.7 mm Cu) were administered at a rate of approx. 50 R per min in air. In each experiment all treated cultures were irradiated simultaneously with the controls at r o o m temperature. Colcemid was added immediately after irradiation to all cultures, which were then reincubated at 37 ° for the 2 or 3 h prior to harvest.
192 For cultures to be used for autoradiography to obtain percent labeled mitosis (PLM) curves tritiated thymidine (New England Nuclear; 6.7 Ci/mM) was added to a final concentration of 1/~Ci per ml of medium and allowed to remain until the cultures were terminated. In the case of lymphocytes, cultures were fixed every hour following addition of the tritiated thymidine up to the 9th hour; fibroblast cultures were fixed at 1.5 h, and then each hour up to 9.5h. The cells were fixed in 3 : 1 methyl alcohol : glacial acetic acid following a 15-min hypotonic treatment with 0.075 M KC1. After washing with fresh fixative, cells were mounted by dropping the suspension into the surface of wet glass slides and then flaming. Slides for autoradiography were dipped in NTB-2 nuclear-track emulsion (Kodak) diluted 1 : 1 with distilled water, dried, exposed for one week and then developed in D-19 developer for 4 min, fixed and allowed to air dry. Slides were stained with 10% Giemsa (Harleco). Aberrations were scored at 800×, following commonly accepted criteria for the various aberration types. Metaphases were scored as labeled in the autoradiographs if there were more than 10 grains over them after subtracting an average background value determined separately for each slide by counting grains over areas not containing any cells. Results A dose--effect curve was obtained for one FA patient's lymphocytes in order to determine a single appropriate X-ray dose for later experiments. With the I 3.25
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Fig. 1. T o t a l c h r o m o s o m a l a b e r r a t i o n y i e l d s in peripheral l y m p h o c y t e s f r o m a F A p a t i e n t ( o p e n circles) and f r o m a n o r m a l c o n t r o l ( h a l f - c l o s e d circles) given various d o s e s o f X-rays in t h e G 2 phase o f the cell c y c l e . T h e single c l o s e d circle r e p r e s e n t s t h e average u m r r i d i a t e d c o n t r o l value for 7 9 2 cells f r o m 4 F A p a t i e n t s (see t e x t ) . Bars i n d i c a t e P o l s s o n errors. 1 0 0 cells w e r e s c o r e d per p o i n t for all o t h e r s a m p l e s .
193
exception of the unirradiated FA culture, 100 cells were scored per point. The results are summarized in Fig. 1. Because the unirradiated F A culture in this experiment failed to yield but a few scoreable metaphases, the point shown is the average value for all of our unirradiated FA lymphocyte cultures. The bars indicate Poisson errors. The lines shown are least-squares linear regressions. On the basis of this preliminary experiment, a "standard dose" of 50 R was adopted for 5 additional experiments with lymphocytes from 3 other patients. The results, including the control and 50-R data from the experiment shown in Fig. 1, are given in Table 1. It will be seen that in spite of some variability in magnitude, in each experiment the yields of both achromatic lesions and chromatid deletions in the F A cells are higher than in the controls. Table 2 gives a summary of the pooled data from all of the lymphocyte experiments. Because of the different sample sizes in two of the experiments, the net increases were calculated by subtracting the number of aberrations expected in a sample the size of the irradiated one on the basis of their frequency in the unirradiated one. The errors were based on Poisson variance. The differences
TABLE
1
CHROMOSOMAL LYMPHOCYTES
ABERRATIONS FROM FANCONI'S
IN IRRADIATED AND UNIRRADIATED PERIPERAL BLOOD ANEMIA PATIENTS AND NORMAL CONTROL INDIVIDUALS
Subject
Irradiation (R)
Number of cells scored
Achromatic lesions
1
FA - LE
None 50
11 100
26 189
2 47
2 36
C - 1
None 50
100 100
6 64
1 11
1 0
FA - KE
None 50
200 200
46 318
22 145
5 89
C - 2
None 50
200 200
29 93
14 28
1 12
FA - KE
None 50
150 150
41 228
14 104
5 4
C - 3
None 50
150 150
11 49
3 18
0 10
FA - BA
None 50
100 100
27 167
13 84
1 8
C - 4
None 50
100 100
11 64
9 37
1 0
FA - LA
None 50
150 150
37 272
10 106
0 10
C - 5
None 50
150 150
18 108
17 43
0 4
FA - KE
None 50
181 231
51 901
4 592
1 31
C - 4
None 50
100 285
14 775
0 437
0 44
2
3
4
5
6
a Includes isochromatid tions were seen.
deletions
and chromatid
interchanges;
Chromatid deletions
Others a
Expt.
no unequivocal
chromosome-type
aberra-
194
TABLE 2 POOLED CHROMOSOMAL ABERRATION YIELDS FROM THE 6 LYMPHOCYTE TION EXPERIMENTS FOR WHICH THE RAW DATA ARE GIVEN IN TABLE 1
G 2 X-IRRADIA-
N e t i n c r e a s e s c a l c u l a t e d b y s u b t r a c t i n g t h e n u m b e r o f e a c h t y p e o f a b e r r a t i o n e x p e c t e d in a s a m p l e t h e size o f t h e i r r a d i a t e d o n e o n the basis o f their f r e q u e n c y in t h e u n i r r a d i a t e d s a m p l e . Number o f cells
Achromahc lesions
Chromatid deletions
Other
Total
FA (4 s u b -
Unirradiated
792
228 2 8 . 8 +- 1.9 %
65 8.2 ± 1 . 0 %
14 1.8 ± 0 . 5 %
307 3 8 . 8 -+ 2 . 2 %
jeers, 6 Expts.)
Irradiated
931
2075 2 2 2 . 9 ± 4.9 %
1078 1 1 5 . 8 +- 3.5 %
178 19.1 + 1 . 4 %
3331 357.8 ± 6.2 %
1807 1 9 4 . 1 + 4.6 %
1002 1 0 7 . 6 ± 3.4 %
162 17.4 + 1.4 %
2971 3 1 9 . 1 + 5.9 %
Net increase Controls (5 s u b -
Um~adiated
800
89 1 1 . 1 + 1.2 %
44 5.5 + 0.8 %
3 0.4 ± 0.2 %
136 1 7 . 0 + 1.5 %
jects, 6 Expts.)
Irtadiated
985
1153 1 1 7 . 1 -+ 3 . 5 %
574 5 8 . 3 -+ 2.4 %
70 7.1 + 0.9 %
1797 1 8 2 . 4 + 4.3 %
1043 1 0 5 . 9 ± 3.3
520 5 2 . 8 ± 2.3 %
66 6.7 + 0 . 8 %
1629 1 6 5 . 4 -+ 4 . 1 %
Net increase
between the irradiated FA and the irradiated control cells are, of course, statistically highly significant. The raw results of our experiments with the 3 FA-fibroblast lines are given in Table 3, and summarized in Table 4. The net increases were calculated as for Table 2. While the aberration yields are lower than in the lymphocyte experiments, very likely because of a longer G2 phase, it can be seen that the fibroblast results correspond qualitatively to those for lymphocytes, and that FA fibroblasts are also characterized by a significantly elevated susceptibility to TABLE 3 CHROMOSOMAL ABERRATIONS IN IRRADIATED AND UNIRRADIATED SKIN-BIOPSY FIBROBLAST CULTURE CELLS FROM 3 FA PATIENTS AND 3 NORMAL CONTROL INDIVIDUALS Expt.
Culture
Irradiation (R)
Number of cells ~ o r e d
Achromatic lesions
Chrom~id dele~ons
Others a
1
FA - LE
0 50
100 100
13 146
7 48
3 10
C-6
0 50
100 100
13 50
9 14
3 0
GM-368
0 50
75 75
16 133
14 49
1 3
C-I0
0 50
75 75
14 24
8 15
0 0
GM-391
0 50
75 75
10 131
8 49
0 4
C-323
0 50
75 75
10 13
4 12
0 0
2
3
a Includes isochromatid deletions and chromatid interchanges; no unequivocal chromosome-type aberrations were seen.
195
TABLE 4 POOLED CHROMOSOMAL ABERRATION YIELDS FROM THE 3 FIBROBLAST G2 X-IRRADIATION E X P E R I M E N T S F O R W H I C H T H E R A W D A T A A R E G I V E N IN T A B L E 3
Number o f cells FA (3 E x p t s . )
Unirradiated
250
I*Tadiated
250
Achromatic lesions
Other
39 1 5 . 6 ± 2.5 %
4 1.6 -+ 0.8 %
410 164.0 +- 8 . 1 %
146 58.4 ± 4.8 %
17 6.8 ± 1.6 %
573 229.2 + 9.6 %
371 1 4 8 . 4 + 7.7 %
107 4 2 . 8 ± 4.1
13 5.2 ± 1.4 %
491 1 9 6 . 4 ± 8.9 %
3 1.2 ± 0.7 %
61 24.4 + 3 . 1 %
39 1 5 . 6 -+ 2.5 %
Net increase Control (3 E x p t s . )
Chromatid deletions
Unirtadiated
250
37 1 4 . 8 ± 2.4 %
21 8.4 ± 1.8 %
Irradiated
250
87 3 4 . 8 + 3.7 %
41 1 6 . 4 ± 2.6 %
50 2 0 . 0 ± 2.8 %
20 8.0 ± 1.8 %
Net increase
Total
0 --
82 3 2 . 8 + 3.6 %
128 51.2 ± 4.5 % 73 2 9 . 2 + 3.4 %
chromosomal breakage by X-rays. Because the yields of both achromatic lesions and chromatid deletions tend to decrease with increasing irradiation-fixation interval following G2 irradiation [30], the observed apparent G2 chromosomal radiosensitivity might result if the FA cells simply take longer to move from the S phase into mitosis. Indeed, such an explanation is suggested by the report of Loughman [15] of difficulty in determining cell-cycle time for lymphocytes of a FA patient because a substantial fraction of the cells would not label with tritiated thymidine, shifting the PLM curve relative to that normal controls in a manner suggestive of a lengthened G2 phase. Fig. 2 shows the PLM curves we obtained in our autoradiographic experiments both with lymphocytes from 2 FA patients and 2 normal
I I I I I CONTROLS (2) LYMPHOCYTES ~, FA (2) LYMPHOCYTES • CONTROL FIBROBLASTS • F.& F I B R O B L A S T S
I
I
I
I
i 7
I 8
J 9
o
co 0 lOG
80 J
< 60 £ w 40 £3 r~
20 0
" I
2
5
4
5 6 TIME (hr)
I0
Fig. 2. P e r c e n t l a b e l e d m i t o s i s c u r v e s f o r p e r i p h e r a l l y m p h o c y t e s ( o p e n s y m b o l s ) a n d fibroblast-cell lines (closed symbols) f r o m FA patients and normal control indiwduals.
196 controls and with a single FA and a control fibroblast-cell line. It may be seen that while the average duration of G2 for the fibroblasts is longer {about 7 h) than that of the lymphocytes (about 5 h), there is little evidence that the PLM curves of FA cells are different from those of normal control cells. Certainly any difference there might be is much too small to account for differences in aberration yield of the magnitude we have observed.
Discussion Our results confirm in one sense the chromosomal sensitivity of Fanconi's anemia cells to ionizing radiation originally reported by Higurachi and Conen [11,12] based upon experiments in which peripheral lymphocytes were irradiated in the Go phase of the cell cycle. Our experiments, however, included only G2 irradiations, and thus do not resolve the conflict between their observation and that of Sasaki and Tonomura [26], who failed to observe substantially increased aberration yields in lymphocytes treated in either the Go or (probably) the S phase. Recently we have made a similar study of lymphocytes from patients with ataxia telangiectasia (AT) treated with X-rays in either Go or G2 [6]. Though we found a G2 chromosomal rasiosensitivity very similar to our present result for FA cells, we were unable to confirm the Go radiosensitivity reported by Higurachi and Conen [ 11,12] for lymphocytes from patients with this desease. It seems possible, then, in agreement with the findings of Sasaki and Tonomura, that FA cells are actually only particularly radiosensitive during the G2 phase of the cell cycle. Whatever the final resolution of the question of chromosomal radiosensitivity during the rest of the cell cycle, our G2 results seem unequivocal. That the phenomenon is a general characteristic of FA is suggested by our similar findings for both lymphocytes and fibroblasts, and for cells from 6 apparently unrelated persons with the disease. Our autoradiographic experiments appear to rule out the possibility that increased aberration yields results simply from a longer G2 phase characterizing FA cells than cells from normal individuals, and suggest that the labeling curve anomaly reported by Loughman [15] for one FA patient is not in fact a general characteristic of FA cells. Unfortunately, the present experiments cannot rule out the possibility that ionizing radiation might simply induce an abnormal delay in the progression of FA cells through G2, thus resulting in the sampling of normal and FA-cell populations irradiated at different stages within the G2 phase at any particular fixation time, and explaining the greater aberration yields in the FA cells. Nevertheless, though additional experiments are clearly needed to settle this point, this possibility seems unlikely to us. Even at AT cells, in which ionizing radiation administered during the G1 (Go) phase of the cell cycle causes markedly longer cell-cycle delays than in normal cells, exhibit little if any delay in their progression to mitosis if irradiated in the G2 [6], though AT cells do exhibit the same G2 chromosomal radiosensitivity phenomenon as FA cells [6,22,34]. The definite increases in chromosomal aberration yields induced in FA cells by G2 X-irradiation are of particular interest with respect to DNA-repair mechanisms and the molecular mechanisms involved in the induction of aberrations by ionizing radiation. If, as reported by Remsen and Cerutti [23] and by Rain-
197
b o w and Howes [21] FA cells are deficient in their ability to repair a form of DNA-base damage induced b y ionizing radiation, then the excess aberrations might be attributed to unrepaired (or partially repaired) lesions of this type, rather than, perhaps, to higher initial amounts of DNA damage. Unfortunately, however, defects in repair of other DNA lesions, including double and single polynucleotide strand breaks, cannot be ruled o u t on the basis of information presently available. Furthermore, the specific deficiency noted by Remsen and Cerutti, involving the cells' ability to excise products of the 5,6-dilhydroxyd i h y d r o t h y m i n e type, was found in only 2 of 4 FA-cell lines tested, and so may n o t be generally characteristic of the disease. The aberrations seen in both the FA and the normal cells in our experiments were all of the chromatid type, and consisted mainly of achromatic lesions and simple chromatid deletions. Only a relatively few isochromatid deletions or chromatid exchanges were seen, as is the normal consequence of irradiation of cells in G2 [30]. Thus the increased yields in the FA cells were largely, at least, in the classes both induced in normal cells by ionizing radiation and arising spontaneously in untreated cells. Several lines of evidence suggest that single polynucleotide strand breaks may appear as achromatic lesions at metaphase [3,32] and double polynucleotide strand breaks must surely at least be involved in chromatid breakage [4,18]. It furthermore seems clear that some single polynucleotide strand breaks must become double-strand breaks and contribute to chromatid breakage [3,5,18]. Thus the observed increases in achromatic lesions and in chromatid deletions in our irradiated FA cells appear consistent with the possibility that FA cells are simply deficient in their ability to repair single polynucleotide strand breaks. Nevertheless, because excision-type repair of damaged DNA bases appears to involve a number of enzymatic steps, and because a discontinuity in the polynucleotide strand under repair must exist during part of the excision process, it is also possible to reconcile the present cytogenetic results equally well with the idea that FA cells are characterized b y a defect in excision repair of damaged bases like that found by Remsen and Cerutti [23]. It would only be required that the defect involve a step after the " n i c k " is made, which could result in a larger number of single-strand breaks being present in FA than in normal cells, and thus, if achromatic lesions and chromatid deletions are in fact attributable to such breaks, in larger yields of these aberration types. The observations both of Remsen and Cerutti and of Rainbow and Howes [21] are consistent with such an idea, since the former measured the appearance of the damaged base in the acid-soluble fraction following treatment of irradiated DNA with cell or nuclear sonicates, while the latter measured host-cell reactivation of irradiated virus. The recent finding of Natarajan and Obe [18] that treatment of Chinese hamster tissue-culture cells irradiated in G2 with a Neurospora endonuclease specific for cleaving single-stranded DNA gave increased yields of chromatidt y p e aberrations observed at metaphase is also consistent, though it also suggests the alternative possibility that FA cells might simply possess more of a similar enzymatic activity than normal cells. Of course it is possible as well that a defect in a step c o m m o n to both damaged-base excision and strand-break repair could also account for all of the observations. It is of interest to compare the results for FA cells with the information
198 available on AT cells, which are in some respects similar in their response to ionizing radiation. Both FA and AT lymphocytes have been reported to be chromosomally radiosensitive to ionizing radiation during the Go phase [11,12] though as already noted we have been unable to confirm this observation for AT lymphocytes [6]. Nevertheless, precisely the same sort of G2 chromosomal radiosensitivity has been found in AT cells irradiated in G2 as we here report for FA cells [ 6 , 2 2 , 3 4 ] . Furthermore, Taylor et al. [34] have demonstrated that Go irradiation of AT lymphocytes results in a small but statistically significant yield of aberration of the chromatid type, an unexpected consequence of Go or G1 irradiations, but one which we have been able to confirm [6]. As with FA cells, a defect in the ability of AT cells to excise DNA-base damage has been reported [19], though the base damage is not of the 5,6-dihydroxydihydrothymine type deficient in at least some FA cells [24]. In addition, it has been reported that AT cells will show a normal ability to repair both single and double polynucleotide strand breaks [ 1 4 , 3 3 , 3 6 ] . It is therefore possible to conclude that the increased G2 aberration yields in irradiated AT cells arise from the base-damage repair defect rather than from a parallel polynucleotidestrand break-repair deficiency, and thus infer that this is at least possible in FA cells as well, even though the type of base damage for which excision appears to be deficient in the t w o diseases is not the same. References 1 Auerbach, A.D., and S.R. Wolman, Susceptiblhty of Fanconi's anaemia fibroblasts to chromosome damage by carcinogens, Nature (London), 261 (1976) 494--496. 2 A u e r b a c h , A . D . , a n d S.R. W o l m a n , C a r c i n o g e n - i n d u c e d c h r o m o s o m e b r e a k a g e m F a n c o n i ' s a n a e m i a h e t e r o z y g o u s cells, N a t u r e ( L o n d o n ) 2 7 1 ( 1 9 7 8 ) 6 9 - - 7 1 . 3 B e n d e r , M . 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