J. Mol. Riol. (1976)
103, 599-610
Critical DNA Damage and Mammalian
Cell Reproduction
H. JOHN BVRKI Physics and th,r Do,rrwr Lahorntory L)ivisio~~ of Ndical University of Calijornia Bwkeley, Calif. U.rS.4. (Received 9 September 1974. and in revised form 16 January
1976)
Synchronous Chinese hamster cells accumulated 1251-induced DNA damage in the G, $ Mt period at 4°C. The position of the lz51 within the nuclear DNA was varied by incorporating [ lz51]iododeoxyuridine at various times during the previous DNA replication period. When the initial cell inactivation efficiency was compared for damage accumulated in various regions of nuclear DNA, it was found that the efficiency of inactivation was least in early replicating DNA, and t’hat it gradually increased, reaching a maximum during the fourth and fifth hours of the six-hour DNA replication period. Because the DNA that replicated during this maximum corresponds to that DNA which later forms centro-
meric and near-centromeric
regions of the chromosomes, damage in centromeric
region
in causing
DNA
may be critical
mammalian
cell inactivation.
1. Introduction The replication of DNA within the mammalian cell nucleus is a highly organized process. It proceeds in three dimensions in such a way that early in the replication (S period-t) a diffuse replication pattern is found in the nucleus, and that this replication pattern becomes peripheral at the end of the S period (Comings & Okada, 1973; Huberman et al., 1973). DNA replication also seems to be related to the state of the chromatin in the nucleus because heterochromatin usually replicates late, while euchromatin, which is used in the production of RNA, usually replicates early (Comings, 1972a; Utakoji & Hsu, 1965), and because several mutations have been mapped in early replicating DNA (Suzuki & Okada. 1975; Aebersold & Burki, 1976). DNA replication order is related to cell differentiation as seen when hamster cells from testes, where the Y chromosome replicates early, are compared to other somatic cells where the Y chromosome replicates late (Utakoji & Hsu, 1965). DNA replication sequence does not change in successive cell divisions, so early replicating DNA is always early replicating (Mueller t Kajiwara, 1966). Specialized functional DNA-like nucleolus-organizer DNA replicates at a specific time (Stambrook. 1974 ; Amaldi et al., 1969). Zn addition, DNA replication patterns appear to be related to the metaphase chromosome banding patterns, because in human cells interband regions always replicate early (Ganner & Evans? 1971). while in hamster cells regions near centromeres usually replicate late (Stubblefield. 1975). The time of replication may also be related t Abbrt:viatIionx used: G, and G,. prr itncl post 11X.4 a,vrrt~hwis periods rtqwctivrly UNA synthesis; M stage, mitosis.
; 8 pet-id,
800
H.
J.
BURKI
t,o the number of repeated sequences in the DNA; in some cases highly repetitious DNA replicates late (Flamm et al., 1971; Bostock & Prescott, 1971a). Our hypothesis is that this replication informat’ion about the organization of the mammalian nucleus might be used to make a functional test of a specific subnuclear region of the DNA using the technique of “molecular suicide” (Ragni & Szybalski, 1962). This method has been extensively applied to procaryotic systems and, to a lesser extent, to mammalian cells to study reproductive death, and repair and damage of DNA (Burki & Okada. 1968.1970; Cleaver ef al.. 1972: Burki, 1974; Painter cf al.. 1974). Until recently t’he choice of a possible isotope to use to damage parts of the mammalian nucleus selectively was primarily limited to tritium (Burki & Okada, 1968, 1970). This isotope has t,wo disadvantages for local damage of nuclear DNA : its range is of the order of 0.5 to 1.0 pm, and it causes damage that is rapidly repaired (Cleaver et al., 1972) so that repair complicates the interpretation of results of suicide experiments. The availability of lz51, especially in the form of [1251]iododeoxyuridine, solved these two problems because the effects are associated primarily with result’s induced by Auger phenomena (Burki et al., 1973), which makes this isotope extraordinarily toxic. Recently it has been shown that much of the DNA damage produced is non-repairable (Painter et aE., 1974) and that [ 1251]dUrd decay is an extremely efficient inducer of chromosome aberrations of the two-break type in Chinese hamster cells (Burki et al., 1976). Thus, 1251 is an isotope that may ha.ve the most useful characteristics for testing the functional importance of intranuclear DNA a,nd chromosome damage. We reported in a preliminary study that the nucleus was not homogeneous with respect to killing induced by [ lz51]dUrd decay, and that the DNA replicated in the second half of the DNA synthesis period seemed to be most important (Burki, 1974).
2. Materials and Methods (a) Cell culture Chinese hamster cells strain V79-s171 were grown in tissue culture in a medium composed of: Puck’s saline F (GIBCO), 500 ml; amino acids (100 x ; BME, GIBCO) (200 mM) 5 ml; penicillin GIBCO) 5 ml ; L-glutamine 5 ml; vitamins (100 x ; BME, (100,000 units/ml) and streptomycin (10,000 pg/ml) 5 ml; CaC1,.2Hz0 (33% w/v) 0.15 ml; fetal calf serum 10% (v/v). These cells were free of plcuro pneumonia-like organisms and wore from a stock of cells kept in liquid nitrogen and protected from freezing damage using a 10% dimethylsulfoxide/media suspension (Burki & Okada, 1968). This cell line is a pseudodiploid cell line with a modal chromosomal number of 23 chromosomes (Burki & Carrano, 1973). (b) Synchrony
method
The method of synchrony which we found finally to be most successfully reproducible was the following: cells grown for 72 h that were in logarithmic growth were subculturod into 12 to 24 4.0~ Saniglas or Duraglass prescription bottles, at a final concn of 3 x lo5 cells/bottle (3 x lo4 cells/ml) and incubated at 37°C for 18 to 19 h before the addition of were Colcimid (GIBCO). Colcimid (0.06 pg/ml) was added for 1 h and t,he bottles then shaken on a platform at‘tached to a wrist-action shaker (Burrel Corp.) at a radius of 24.5 cm and a setting of 2.5 which corresponds to a horizontal displacement of &2 cm. The mitotic index of this mitotically detached population was 75% or more. This mitotic population was centrifuged at 3000 revs/min for 15 min (150 g), the Colcimid medium decanted, and the cells added to plastic culture dishes containing 1 mMhydroxyurea in normal growth medium. After 7 h in hydroxyurea the attached cells at the G,-S interface
CRITICAL
DNA
DAMAGE
601
in t,hc cell cycle (see Results) were released from the hydroxyurea block by washing with warm (37°C) Puck’s saline F (GIBCO) solution; t,hey were then incubated fresh warm media containing 10e6 M-iododeoxyuridino.
(c) Labeling
procedure
and determination
of plating
twice with
eficiemcy
Control populations were given iododeoxyuridine alone. Then 6 h after the release from the hydroxyurea block tlir flasks containing the cells were placed in a refrigerator at 4°C. These cells were then stored in the refrigerator in closed plastic flasks (Falcon no. 3012) for up to 4 days. At various times during the storage at 4°C the cells were removed from the refrigerator, trypsinized, and then plating efficiency was determined by the usual methods. Control survival for these experiments was 355, t- Bn,, and there was almost, no decline in this plating efficiency for 3 days at 4°C. Other cells were permitted to incorporate [ 1251]dUrd at a variety of times in the initial DNA synthesis period after the release from the hydroxyurea block. The formats of the various experiments are given in Results. After incorporating this isot,ope the cultrues were washed as described above and reincubatod wit,11 fresh medium containing a chase of 1W6 ,M-deoxyuridine. Most experiments were designed so that less than 2 decays of [1251]dUrd occurred during the incorporation period. In certain experiments higher amounts of radioa,ctivity incorporation were used, but in t,hese cases the purpose was to demonstrate the hoterogeneity of labeling which occurred in the population of cells and led to a tail on the survival curve. These latter experiments were not, used for internuclear comparisons of effectiveness of [‘251]DNA decays.
(d) Determination
of
radioactivity
autoradiographic
per cell am1 procedure
A tutal of from lo5 to lo6 cells was centrifuged down to a pellet in a plastic test tube and washed with ice-cold 10% trichloroacetic acid. The supernatant was then removed and the pellets and supernatant counted separately using an automatic well crystal gamma spectrometer calibrated with an lzsI counting standard (New England Nuclear no. NES-211s). The radioactivity in the acid-soluble fraction was always 20/, or less in t,hese experiments. Other experiments not reported flere have demonstrated that almost all the rest of the radioactivity is in the DNA (Painter et al., 1974). The specific activities used in these experiments varied from 1O- * to 10-s $?i/mmol and the final disintegration rate was 0.06 to 1.0 disints/h per cell. In order to prepare autoradiograms, harvested cells were dropped onto slides and airdried. In a darkroom the slides were dipped in Kodak nuclear track emulsion no. ? and stored using silica gel to help dry the film. Development was done with Dektol (Kodak). The slides were stained using Giemsa stain at pH 6.8.
3. Results (a) Toxicity
of IdUrd
and [1251]dUrd in synchronous
asynchronous
and
cells at 37°C
The survival curve of cells that were synchronized by the mitotic shake-off t.echnique without a subsequent hydroxyurea block is shown in Figure l(a). These cells were labeled with different specific activities of [1251]dUrd at low6 M-IdUrd for 16 hours in order to compare the results with those found using asynchronous logarithmically growing Chinese hamster cells (Fig. l(b)). The synchronous data (given by the broken line) are very similar to the asynchronous results. This suggests that there was no selection in the synchrony technique for especially sensitive mammalian cells. Figure 1 (b) also shows the toxicity of unlabeled IdUrd in this cell line. The toxic level for these experimental conditions is approximately 10 -5 M-IdUrd. Thus, 10e6 MIdUrd was used to avoid toxicity problems. IdUrd toxicity is important in these
H. Fmal disints/h
[ ‘251]dUrd
(@I
J.
per cell
/ml)
(a) FIG.
BURKI lododeoxyuridine
pCI/mlof
concn (M)
[‘25 IldUrd +IO-6M-dUrd (b)
1. (a) The
survival of cells synchronized by mitotic selection and then permitted to in. for 16 h at 37°C and then plated to determine the percentage of cells able to form visible colonies. On the upper abcissa is the final disints/h per cell of lz51 in the cell DNA and on the lower abcissa is given the t#otal activity of [ ‘251]dUrd used in the media with a carrier of 10-s ix-IdUrd. (In these experiments spec. act. = total act/lo-s rnM per ml.) (b) The survival of asynchronous hamster cells after incorporation of [1251]dUrd for 16 h at 37°C and then plated to determine the percentage of cells able to form visible colonies (a). This data makes use of the lower abcissa where the log of the total activity of the [‘e51]dUrd is given for convenience. The broken line is the data shown in (a). The survival of asynchronous hamster cells after incorporation of cold iododeoxyuridine for 16 h at various concentrations (0).
corporate [ ‘251]dUrd
experiments because, to ensure uniform labeling of the DNA, very low specific activities of [1251]dUrd must be used. The use of high specific activity [lz51]dUrd leads to uneven labeling of the DNA because of exhaustion of [1251]dUrd in the medium by the cells (Burki & Okada, 1970). (b) Synchrony
before labeling,
at cooling and after storage at 4°C
In order to evaluate the consequence of [lz51]DNA decays at different regions within the nucleus at a fixed time in the cell cycle, it was important to determine the ,&ate of the cells in the cell cycle at various times during the experiment (Fig. 2). Figure 2(a) shows the percentage of mitotic cells and the percentage of labeled cells (15min pulse of [3H]thymidine (13.7 Ci/mmol, 0.1 ,&/ml)) at various times during and after the hydroxyurea block. The mitotic population dropped from 75yo at time zero to 0% 30 minutes later. There appeared to be a small persistent fraction of cells (5 to 10%) in DNA synthesis during the hydroxyurea block. When the hydroxyurea block was released, there was a rapid entrance into DNA synthesis while the mitotic index remained at zero. In order to determine the approximate position of the cells within the cycle at the time of cooling to 4°C the data shown in Figure 2(b) and (c) were obtained. In Figure 2(b) a very late S-phase cell cycle marker was used. Because it is well known that Chinese hamster cell lines have a very resistant period to X-ray radiation inactivation at the end of the DNA synthesis period, cells were irradiated at various times after release from the hydroxyurea block with 1150 rad of X-rays (145 kV; 153 rad/ min) (Burki & Carrsno, 1973). The data indicate that the majority of cells in this
CRITICAL M
G,,, +
IO v) = s 0
8-t
I
L)Nd
hydroxy urea block I
SI I
I 4; I I I 2 -1
a,
A ABlock --.-..-----•
I O-’ @~--‘-----l~----~ 2 4 0 Time afte: mltotic
SI
T : : s -0 g, : P
.I I
1 (a) I
6-1
5 kl a
6
release -1 IO
8
cell detachment
a
(h)
%,I
Time of cooling
(b)
- 100
1
I I
z E 6 0) $
IO=
603
DAMAGE
/“\,’ 0
I60 \ ?,
0.1 i
5
i 0 “Lo
001. 0
1 I 2
I 4
Time ofter hydroxyureo removal (h)
I 6
B
0
2
4
6
8
I
Time after release from hydroxyureo (h)
FIG. 2. (a) The percentage of mitotic cells (a), and the percentage of the synchronized cells incorporating high specific activity tritiated thymidine (A), is a function of time after initial selection of mitotic cells which are then grown in a media containing hydroxyurea for 7 h and then washed and grown in fresh media at 37’C. (b) The percentage survival of the synchronized cell population exposed to 1150 rads of roentgen radiation at different times after the initial hydroxyurea block is removed. (c) The number of colonies per 1000 cells after a second exposure of hydroxyurea, as a function of time after release from the first hydroxyurea block for different experimental formats. (A) No IdUrd; (0) 10-s M-IdUrd and washing; (;?) 10-s M-IdUrd fir&half S period; (0) 10-s M-TdUrd second-half of the S period.
synchronous population reached their resistant stage at five hours. The data given in Figure 2(c) can be used to indicate the time of entrance into the G, period as a result of the different experimental formats. The different experimental procedures did not effect the time of entrance into the G, period. After hamster cells were placed in the refrigerator the progress through the cell cycle rapidly slowed down and macromolecular synthesis was reduced to nearly zero (Nelson et al., 1971). During four days storage at 4°C the cells progressed slowly toward the M stage (Burki, unpublished observations).
H.
604
(c) Killing
eficiency
J.
BURKI
of [‘251]DNA disintegrations
in different
intranuclear
regions
The results of a typical experiment are given in Figure 3. The slope of the survival curve was zero for controls and for IdUrd alone, and increased as the t,ota,l activity of the 1251 in the medium increased. However? the highest total activity experiments showed a resistant component on the survival curve at longer times. This resistant component was always seen and varied with the highest amount of lz51 used in the experiments. When data of the type given in Figure 2 were related to the total number of 1251 decays in DNA, the results in Figure 4 were found. Because each experiment was a composite of three dose rates, and the combined data fell on a straight line (Fig. 4), dose rate effects are not reflected in these experiments. The survival curves shown in Figures 3 and 4 are at least biphasic, including the curves for cells labeled for the
0
10
20
30
40
50
60
70
Time of storage at 4°C in growth media (h)
FIG. 3. The measured percentage survival ment at 4 different levels. (0) Just 10e6 [‘26I]dUrd (0) as a function of the number
for cells incorporating iwIdUrd; and either of hours at 4°C.
in a typical experi[ 1251]dUrd low, medium, or high levels of
complete DNA synthesis period (Fig. 4(b)). The initial slope of the survival curve for Figure 4(a) (first half of the S period) was O-031, corresponding to an initial Do7 of 32.3 decays, whereas the cells labeled for the complete S period had an initial slope of 0.042, corresponding to a D,, of 23.8 decays. The steepest initial slope is given in Figure 4(c) for the cells that incorporated [ 1251]dUrd in the second half of the DNA synthesis period, which has a slope of 0.063 or a D,, of 15.9 decays. In a preliminary study (Burki, 1974) the initial slope difference between [ 1251]dUrd incorporated during early S and late S period was measured for small amounts of [1251]dUrd. In that study there was still a possibility that resistant components of the complex survival curve might influence the observed differences in the efficiencies of inactivation. For this reason, experiments were conducted with much higher amounts of [1251]dUrd to the number of decays to reduce the survival by l/e or e-l t Abbreviations used : D,, (e = 2.7182) in the linear portion of the survival curve (used when the survival curve has a of decays to kill 63% of the cells in a simple exponential survival shoulder); D,,, the numljer curve.
CRITICAL
DNS
005
DAMAGE
I 200
I 100
0
100
200 No. of ‘*’
300 I decays
400
600
000
3c
IOC
m ONA
4. (a) Survival curve for synchronized cells stored at 4’C in the Gz period after incorporating [lz51]dUrd into the DNA replicated in the first half of the S period. Composite of several individual experiments (0); individual experimental results ( 0). Error bars are either standard error of the mean or standard deviations. (b) Survival curves for synchronized cells stored at 4°C in the Gz period after incorporating [‘251]dUrd unifilarily into the total replicated DNS. Results from two experiments. Symbols as in (a). (c) Survival curves for synchronized cells stored at 4°C’ in the GZ period after incorporating [‘251]dUrd into the DNA replicated in the second half of the S period. Composite results of 6 experiments (0). Two other series of experiments at higher dose levels (0) and ( 3). FIG.
investigate the more resistant components of the survival curves. The results show that there were resistant components on all of the observed survival curves, including curves for cells that had been labeled for one complete round of DNA replication (Fig. 4). These resistant components permit a resolution of the survival curves into at, least two components in all three cases shown in Figure 4. For the first half of the S period the resolved initial component was 30 decays; for the unifilar labeling the initial component was 15 decays ; and for the second half of the S period the initial component
606
H.
J.
BURKl
was 11 decays. The important point to note is that the differences continued to exist even when these complex survival curves were resolved. However, for simplicity, we elected to take the uncorrected initial slope of the survival curves. The survival curve “resistant, components” have another important significance in these studies. They could be due either to a small fraction of the population of cells that are extremely resistant to [ lz51 ] dUrd decays in cell DNA, or to heterogeneous distribution of label among cells. These two possibilities were distinguished by autoradiography. If there were slow growing cells in t’he populat’ion, or cells which incorporated less [ 1251]dUrd than other cells, the histograms of the grain distribution should be quite widespread. If there was a small variation in the grains per cell the survival curve “tails” would be due to extremely resistant cells. Figure 5(a) shows the histogram of the grain distribution for cells labeled in the first half of the S period. while Figure 5(b) shows the data for t.he second half. In both cases the distribution of grains is rather large and there is a fraction of cells which is lightly labeled. This fraction explains the resistant component of the survival curves. The lightly labeled fractions seen in Figure 5(a) and (b) were similar. In autoradiographs of cells that’ were exposed for longer times there always existed a Sq/, or smaller fraction of very lightly labeled cells. The reason for this heterogeneous labeling is not clear. WC also noted that the film efficiency found in the first half of the S period \vas I 1’:; compared to 14% in the second half, a difference which probably reflect’s the peripheral distribution of DNA replication in hamster cells in the second half of the S period (Huberman et al., 1973 ; Comings & Okada, 1973). After confirming that the resistant component of the survival curve was due to lightly labeled cells in all the labeling procedures, 25 experiments were performed with various experimental formats to determine the efficiency of inactivation of cells for
Second half of S period
FIG? half of S permd
241
0
10 20 30 40 50 60 70 No. of silver (a)
grains (b)
FIQ. 5. Histogram of the number of silver grains per cell after the synchronous population had incorporated [lasI]dUrd into the DNA synthesis period in the first half of the DNA synthesis period (a); and the second half of the DNA synthesis period (b).
CRITICAL
DNA
DAMAGE
ties
Midtime of labeling period in S period (h) Fm. 6. The initial cell inactivation efficienoyt per izsI disintegration into time of the labeling period in which [ ‘a51]dUrd was incorporated labeled from 3 to 6 h in the second half of the DNA synthesis period the t Delined as 1/iD3, = i.e., where ,D,, equals the dose to kill 63% portion of the cell survival curve.
as a function of the midcell DNA (e.g., for oells midtime is at 4.6 h). of t,he cells in the initial
damage accumulated in various DNA regions. A summary of the results is given in Figure 6. These results show that the killing efficiency of [lz51]DNA depended on the site of the lz51 decays within the DNA. The initial D,, was 62.5 decays for the DNA labeled in the first quarter of the DNA synthesis period, 40 for the first third, 32 for the first half, 28 for the third quarter, 16 for the second half or two-thirds and 18 for DNA synthesized in the last quarter. The killing efficiency appeared to be at a maximum between the fourth and the fifth hour of the S period, although the results for the last quarter of the S period were not statistically different from those at maximum efficiency.
4. Discussion (a) The critical
DNA
damage hypothesis
The initial cell inactivation efficiency per lZ51 decay changed depending on what part of the DNA was labeled and was damaged by the decay of this isotope. In those experiments in which Chinese hamster cells were stored at 4°C at the G, + M stage of the cell cycle there appears to be a maximum sensitivity for cells which receive damage in the DNA which replicates about three-quarters of the way through the S period. This damage to late replicating DNA is critical in preventing the reproduction of the Chinese hamster cell. The most likely reason why this damage is critical is because it leads to lethal chromosome aberrations. The recent finding that [1251]dUrd decays in DNA are efficient inducers of dicentric and ring chromosomes, with 35 lz51 decays leading to one dicentric or ring chromosome per cell on the average, is important in this regard (Burki et al.. 1976). Since this dose of 35 lz51 decavs , is similar t’o the D,, for hamster
608
H.
J.
BURKI
cells, this suggests that the lethal event caused by lz51 decay is the induction of dicentrics or ring chromosomes. Recently, Hughes et al. (1976) have shown that lz51 decays induce chromatid aberrations in G, + M cells at the sites where these decays occur. Thus, our hypothesis is that regions exist in mammalian cell DNA where 125I damage leads to chromosome aberrations and to reproductive death more efficiently than in other regions, and that these critical regions are located in the DNA that replicates three-quarters of the way through the S period in Chinese hamster cells. There are many data to support the hypothesis that chromosome aberrations induced by ionizing radiation are non-random in many different systems, including Vicia $&a (Revell, 1953), rat kangaroo (Van Stennis et al., 1974), and human cells (Seabright, 1973; Caspersson et al., 1972). There appears to be evidence that the heterochromatic regions of the chromosomes are the regions where chromosome aberrations occur in these systems. Because most of the heterochromatic parts of chromosomes contain DNA that replicates late, the results reported here confirm these earlier observations. Another way to explain that 125I decays lead to reproductive death with different efficiencies in different parts of the cell DNA, is to propose that some regions of the interphase chromatin are less accessible to repair of 1251-induced damage than other regions. This hypothesis would predict that the reason for the differences depicted in Figure 6 is that the DNA that replicates late in the DNA synthesis period is less accessible to repair and may contain a higher percentage of non-repairable damage. This repair hypothesis is not incompatible with the critical DNA damage hypothesis proposed above because critical chromosome damage still could be the reason for cell inactivation, although it could result from lack of repair. (b) Characteristics
of lute replicating
DNA irb mammalian
cells
Generally the hamster DNA that replicates late in the DNA synthesis period is heterochromatic (Comings, 1972b), although there is still the possibility that certain critical messengers necessary for mitosis or some other vital function also replicate late. It is known that parts of chromosomes replicate at different times in the S phase. In the mouse, for example, centromeric heterochromatin replicates in the third quarter of the S period (Bostock & Prescott, 1971b). In the hamster, centromeric heterochromatin is replicated at approximately four to five hours before mitosis of chromosomes 4 and 5, but certain other chromosomes replicate their centromeres at a later time (Hsu, 1964). Recently, Stubblefield (1975) has shown that most of the regions near the centromeres of hamster chromosomes contain DNA that replicates in the last third of the DNA synthesis period. We can infer from the results of Stubblefield (1975) and others that our proposed critical damage region is associated with the regions of DNA that form the centromeric and near-centromeric regions of chromosomes. Much of the late-replicating DNA replicates in the periphery of the nucleus, near the nuclear membrane (Huberman et al., 1973 ; Comings & Okada, 1973). The fact that this region within the nucleus may be critical has also been suggested by electron penetration experiments (Zermeno & Cole, 1969), where a thin shell critical region including the nuclear membrane has been proposed. This region must contain late replicating centromeric or near-centromeric DNA
CRITICAL
DNA
(c) Other consequences of the critical
609
DAMAGE
DNA
damage hypothesis
One of the striking relationships between the killing efficiency and time of labeling (Wig. 6) is the similarity of the shape of the curve to the shape of the survival curve for Chinese hamster cells exposed to a fixed dose of ionizing radiation during the S period (Bird & Burki, 1975). The two sets of data, although from completely different types of experiments, might be related if one assumes that for replication the critical DNA must be made more diffuse than its normal condensed state. Thus the maximum resistance to ionizing radiation that occurs just before the end of the S period may be related to the fact that critical near-centromeric and centromeric DNA is diffuse and heing replicated at that point. With low linear energy transfer radiation such as S-rays or tritium, much of whose damage might be repaired if there was access to the site before chromosome aberrations are formed, this critical DNA would have the greatest possibility of being repaired during the part of the cell cycle when it was most diffuse. This is similar to the hypothesis of Dewey et al. (1972) formed from their observation that the differential sensitivity to radiation during the cell cycle is greatly diminished by exposure of cells to radiation in a state where all the chromatin was forced to remain more condensed. They found that cell-cycle differences in radiation sensitivity were reduced by hypertonic treatment. The most pronounced effect was to sensitize cells of the late S period. Another consequence of the possible existence of critical DNA damage regions in mammalian cells is that these critical regions might be replicated at different times in t>he S period for different species, and they might be a larger or smaller fraction of the total cell DNA for different species. For example, if mouse satellite DNA was whcrc> critical DNA damage occurred, this might explain the greater radiosensitivity of some neer-diploid cells such as L5178Y cells (Burki et al., 1973), and might also partially explain why t.he response t’o ionizing radiation is different in the mouse during t’he cell a?-cle and whp it is not the same as seen in human and hamster cells. TIw competent technical assistance of Mrs Catherine Lathrop in this work is greatly appreciated. Manuscript preparation was by MS Mary Graham. This investigation was supported by the National Institutes of Health (grant CA-14310) and a grant from the ITT.S. b:rlcr~~
Research
and Development
Administration.
REFERENCES Aebersold, P. 8.x Burki, H. J. (1976). Mutat. Res. 40, 63-66. Amaldi, F., Giacomoni, D. & Zito-Bignami, R. (1969). Eur. J. Biochem. 11, 419-424. Bird, R. & Burki, H. J. (1975). Int. J. Radiat. Biol. 27, 105-120. Bostock. C. M. & Prescott, D. M. (1971a). Eq~t. Cell Res. 64, 267-274. Bostock, C. M. & Prescott, D. M. (1971b). Expt. CeZZ Res. 64, 481--484. Burki. H. J. (1974). Expt. Cell Res. 87, 277-280. Burki. H. J. & Carrano, A. V. (1973). Mutut. Res. 17, 277-282. Burki. H. J. & Okada, S. (1968). Biophys. J. 8, 445-456. Bnrki. H. J. &. Okada, S. (1970). Rudiut. Res. 41, 409 424. Burki. H. J., Roots, R., Feinendegen, L. E. & Bond, V. P. (1973). Id. .I. Rudiat. B,ioZ.
24, 363-375. Burki.
H. .J., Koch, C. & Wolff, S. (1976). C urrent Topics in Radiation Research Quarterly, in the press. Caspersson, T., Haglund, U., Lindell, B. & Zech, L. (1972). Expt. Cell Res. 75, 541-543. Cleavcsr, J. E., Thomas, G. H. & Burki, H. J. (1972). &ience, 177, 996-998. Comings, D. E. (1972a). Expt. Cell Res. 71, 106-112. Comings, D. E. (19723). Expt. Cell Res. 74, 383-390.
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BURKI
Comings, D. E. & Okada, T. A. (1973). J. Mol. Biol. 75, 609-618. Dewey, W. C., Noel, J. S. & Dettor, C. M. (1972). Radiat. Res. 52, 373-394. Flamm, W. G., Bernheim, N. J. & Brubaker, P. E. (1971). Expt. GeZZ Res. 64, 97-104. Ganner, E. & Evans, H. J. (1971). Ghromosoma (Berlin), 35, 326-341. Hsu, T. C. (1964). J. CeEZ Biol. 23, 53-62. Huberman, J. A., Tsai, A. & Deich, R. A. (1973). Nature (London), 241, 32.-36. Hughes, W. L., Weinblatt, A. & Prensky, W. (1976). Current Totics in Radiation Research Quarterly, in the press. Mueller, G. C. & Kajiwara, K. (1966). Biochim. Biophys. Acta, 114, 108-115. Nelson, R. J., Kruuv, J., Koch, C. J. & Fray, H. E. (1971). Expot. Cell Res. 68, 247-252. Painter, R., Young, B. & Burki, H. J. (1974). Proc. Nat. Acad. Sci., U.S.A. 71, 4836-4838. Ragni, G. & Szybalski, W. (1962). J. Mol. BioZ. 4, 338-346. Revell, S. H. (1953). Hereditus (suppZ.), 6, 107-124. Seabright, M. (1973). Chromosoma (Berlin), 40, 333.-346. Stambrook, P. J. (1974). J. Mol. BioZ. 82, 303-313. Stubblefield, E. (1975). Chromosoma (Berlin), 53, 209-221. Suzuki, N. & Okada, S. (1975). Mutat. Res. 30, 111-116. Utakoji, T. & Hsu, T. C. (1965). Cytogenics, 4, 295-299. Van Stennis, H., Tuscany, R. & Leigh, B. (1974). Mutat. Res. 23, 223-228. Zermeno, A. & Cole, A. (1969). Radiat. Res. 39, 669-684.
103, 599-610
Critical DNA Damage and Mammalian
Cell Reproduction
H. JOHN BVRKI Physics and th,r Do,rrwr Lahorntory L)ivisio~~ of Ndical University of Calijornia Bwkeley, Calif. U.rS.4. (Received 9 September 1974. and in revised form 16 January
1976)
Synchronous Chinese hamster cells accumulated 1251-induced DNA damage in the G, $ Mt period at 4°C. The position of the lz51 within the nuclear DNA was varied by incorporating [ lz51]iododeoxyuridine at various times during the previous DNA replication period. When the initial cell inactivation efficiency was compared for damage accumulated in various regions of nuclear DNA, it was found that the efficiency of inactivation was least in early replicating DNA, and t’hat it gradually increased, reaching a maximum during the fourth and fifth hours of the six-hour DNA replication period. Because the DNA that replicated during this maximum corresponds to that DNA which later forms centro-
meric and near-centromeric
regions of the chromosomes, damage in centromeric
region
in causing
DNA
may be critical
mammalian
cell inactivation.
1. Introduction The replication of DNA within the mammalian cell nucleus is a highly organized process. It proceeds in three dimensions in such a way that early in the replication (S period-t) a diffuse replication pattern is found in the nucleus, and that this replication pattern becomes peripheral at the end of the S period (Comings & Okada, 1973; Huberman et al., 1973). DNA replication also seems to be related to the state of the chromatin in the nucleus because heterochromatin usually replicates late, while euchromatin, which is used in the production of RNA, usually replicates early (Comings, 1972a; Utakoji & Hsu, 1965), and because several mutations have been mapped in early replicating DNA (Suzuki & Okada. 1975; Aebersold & Burki, 1976). DNA replication order is related to cell differentiation as seen when hamster cells from testes, where the Y chromosome replicates early, are compared to other somatic cells where the Y chromosome replicates late (Utakoji & Hsu, 1965). DNA replication sequence does not change in successive cell divisions, so early replicating DNA is always early replicating (Mueller t Kajiwara, 1966). Specialized functional DNA-like nucleolus-organizer DNA replicates at a specific time (Stambrook. 1974 ; Amaldi et al., 1969). Zn addition, DNA replication patterns appear to be related to the metaphase chromosome banding patterns, because in human cells interband regions always replicate early (Ganner & Evans? 1971). while in hamster cells regions near centromeres usually replicate late (Stubblefield. 1975). The time of replication may also be related t Abbrt:viatIionx used: G, and G,. prr itncl post 11X.4 a,vrrt~hwis periods rtqwctivrly UNA synthesis; M stage, mitosis.
; 8 pet-id,
800
H.
J.
BURKI
t,o the number of repeated sequences in the DNA; in some cases highly repetitious DNA replicates late (Flamm et al., 1971; Bostock & Prescott, 1971a). Our hypothesis is that this replication informat’ion about the organization of the mammalian nucleus might be used to make a functional test of a specific subnuclear region of the DNA using the technique of “molecular suicide” (Ragni & Szybalski, 1962). This method has been extensively applied to procaryotic systems and, to a lesser extent, to mammalian cells to study reproductive death, and repair and damage of DNA (Burki & Okada. 1968.1970; Cleaver ef al.. 1972: Burki, 1974; Painter cf al.. 1974). Until recently t’he choice of a possible isotope to use to damage parts of the mammalian nucleus selectively was primarily limited to tritium (Burki & Okada, 1968, 1970). This isotope has t,wo disadvantages for local damage of nuclear DNA : its range is of the order of 0.5 to 1.0 pm, and it causes damage that is rapidly repaired (Cleaver et al., 1972) so that repair complicates the interpretation of results of suicide experiments. The availability of lz51, especially in the form of [1251]iododeoxyuridine, solved these two problems because the effects are associated primarily with result’s induced by Auger phenomena (Burki et al., 1973), which makes this isotope extraordinarily toxic. Recently it has been shown that much of the DNA damage produced is non-repairable (Painter et aE., 1974) and that [ 1251]dUrd decay is an extremely efficient inducer of chromosome aberrations of the two-break type in Chinese hamster cells (Burki et al., 1976). Thus, 1251 is an isotope that may ha.ve the most useful characteristics for testing the functional importance of intranuclear DNA a,nd chromosome damage. We reported in a preliminary study that the nucleus was not homogeneous with respect to killing induced by [ lz51]dUrd decay, and that the DNA replicated in the second half of the DNA synthesis period seemed to be most important (Burki, 1974).
2. Materials and Methods (a) Cell culture Chinese hamster cells strain V79-s171 were grown in tissue culture in a medium composed of: Puck’s saline F (GIBCO), 500 ml; amino acids (100 x ; BME, GIBCO) (200 mM) 5 ml; penicillin GIBCO) 5 ml ; L-glutamine 5 ml; vitamins (100 x ; BME, (100,000 units/ml) and streptomycin (10,000 pg/ml) 5 ml; CaC1,.2Hz0 (33% w/v) 0.15 ml; fetal calf serum 10% (v/v). These cells were free of plcuro pneumonia-like organisms and wore from a stock of cells kept in liquid nitrogen and protected from freezing damage using a 10% dimethylsulfoxide/media suspension (Burki & Okada, 1968). This cell line is a pseudodiploid cell line with a modal chromosomal number of 23 chromosomes (Burki & Carrano, 1973). (b) Synchrony
method
The method of synchrony which we found finally to be most successfully reproducible was the following: cells grown for 72 h that were in logarithmic growth were subculturod into 12 to 24 4.0~ Saniglas or Duraglass prescription bottles, at a final concn of 3 x lo5 cells/bottle (3 x lo4 cells/ml) and incubated at 37°C for 18 to 19 h before the addition of were Colcimid (GIBCO). Colcimid (0.06 pg/ml) was added for 1 h and t,he bottles then shaken on a platform at‘tached to a wrist-action shaker (Burrel Corp.) at a radius of 24.5 cm and a setting of 2.5 which corresponds to a horizontal displacement of &2 cm. The mitotic index of this mitotically detached population was 75% or more. This mitotic population was centrifuged at 3000 revs/min for 15 min (150 g), the Colcimid medium decanted, and the cells added to plastic culture dishes containing 1 mMhydroxyurea in normal growth medium. After 7 h in hydroxyurea the attached cells at the G,-S interface
CRITICAL
DNA
DAMAGE
601
in t,hc cell cycle (see Results) were released from the hydroxyurea block by washing with warm (37°C) Puck’s saline F (GIBCO) solution; t,hey were then incubated fresh warm media containing 10e6 M-iododeoxyuridino.
(c) Labeling
procedure
and determination
of plating
twice with
eficiemcy
Control populations were given iododeoxyuridine alone. Then 6 h after the release from the hydroxyurea block tlir flasks containing the cells were placed in a refrigerator at 4°C. These cells were then stored in the refrigerator in closed plastic flasks (Falcon no. 3012) for up to 4 days. At various times during the storage at 4°C the cells were removed from the refrigerator, trypsinized, and then plating efficiency was determined by the usual methods. Control survival for these experiments was 355, t- Bn,, and there was almost, no decline in this plating efficiency for 3 days at 4°C. Other cells were permitted to incorporate [ 1251]dUrd at a variety of times in the initial DNA synthesis period after the release from the hydroxyurea block. The formats of the various experiments are given in Results. After incorporating this isot,ope the cultrues were washed as described above and reincubatod wit,11 fresh medium containing a chase of 1W6 ,M-deoxyuridine. Most experiments were designed so that less than 2 decays of [1251]dUrd occurred during the incorporation period. In certain experiments higher amounts of radioa,ctivity incorporation were used, but in t,hese cases the purpose was to demonstrate the hoterogeneity of labeling which occurred in the population of cells and led to a tail on the survival curve. These latter experiments were not, used for internuclear comparisons of effectiveness of [‘251]DNA decays.
(d) Determination
of
radioactivity
autoradiographic
per cell am1 procedure
A tutal of from lo5 to lo6 cells was centrifuged down to a pellet in a plastic test tube and washed with ice-cold 10% trichloroacetic acid. The supernatant was then removed and the pellets and supernatant counted separately using an automatic well crystal gamma spectrometer calibrated with an lzsI counting standard (New England Nuclear no. NES-211s). The radioactivity in the acid-soluble fraction was always 20/, or less in t,hese experiments. Other experiments not reported flere have demonstrated that almost all the rest of the radioactivity is in the DNA (Painter et al., 1974). The specific activities used in these experiments varied from 1O- * to 10-s $?i/mmol and the final disintegration rate was 0.06 to 1.0 disints/h per cell. In order to prepare autoradiograms, harvested cells were dropped onto slides and airdried. In a darkroom the slides were dipped in Kodak nuclear track emulsion no. ? and stored using silica gel to help dry the film. Development was done with Dektol (Kodak). The slides were stained using Giemsa stain at pH 6.8.
3. Results (a) Toxicity
of IdUrd
and [1251]dUrd in synchronous
asynchronous
and
cells at 37°C
The survival curve of cells that were synchronized by the mitotic shake-off t.echnique without a subsequent hydroxyurea block is shown in Figure l(a). These cells were labeled with different specific activities of [1251]dUrd at low6 M-IdUrd for 16 hours in order to compare the results with those found using asynchronous logarithmically growing Chinese hamster cells (Fig. l(b)). The synchronous data (given by the broken line) are very similar to the asynchronous results. This suggests that there was no selection in the synchrony technique for especially sensitive mammalian cells. Figure 1 (b) also shows the toxicity of unlabeled IdUrd in this cell line. The toxic level for these experimental conditions is approximately 10 -5 M-IdUrd. Thus, 10e6 MIdUrd was used to avoid toxicity problems. IdUrd toxicity is important in these
H. Fmal disints/h
[ ‘251]dUrd
(@I
J.
per cell
/ml)
(a) FIG.
BURKI lododeoxyuridine
pCI/mlof
concn (M)
[‘25 IldUrd +IO-6M-dUrd (b)
1. (a) The
survival of cells synchronized by mitotic selection and then permitted to in. for 16 h at 37°C and then plated to determine the percentage of cells able to form visible colonies. On the upper abcissa is the final disints/h per cell of lz51 in the cell DNA and on the lower abcissa is given the t#otal activity of [ ‘251]dUrd used in the media with a carrier of 10-s ix-IdUrd. (In these experiments spec. act. = total act/lo-s rnM per ml.) (b) The survival of asynchronous hamster cells after incorporation of [1251]dUrd for 16 h at 37°C and then plated to determine the percentage of cells able to form visible colonies (a). This data makes use of the lower abcissa where the log of the total activity of the [‘e51]dUrd is given for convenience. The broken line is the data shown in (a). The survival of asynchronous hamster cells after incorporation of cold iododeoxyuridine for 16 h at various concentrations (0).
corporate [ ‘251]dUrd
experiments because, to ensure uniform labeling of the DNA, very low specific activities of [1251]dUrd must be used. The use of high specific activity [lz51]dUrd leads to uneven labeling of the DNA because of exhaustion of [1251]dUrd in the medium by the cells (Burki & Okada, 1970). (b) Synchrony
before labeling,
at cooling and after storage at 4°C
In order to evaluate the consequence of [lz51]DNA decays at different regions within the nucleus at a fixed time in the cell cycle, it was important to determine the ,&ate of the cells in the cell cycle at various times during the experiment (Fig. 2). Figure 2(a) shows the percentage of mitotic cells and the percentage of labeled cells (15min pulse of [3H]thymidine (13.7 Ci/mmol, 0.1 ,&/ml)) at various times during and after the hydroxyurea block. The mitotic population dropped from 75yo at time zero to 0% 30 minutes later. There appeared to be a small persistent fraction of cells (5 to 10%) in DNA synthesis during the hydroxyurea block. When the hydroxyurea block was released, there was a rapid entrance into DNA synthesis while the mitotic index remained at zero. In order to determine the approximate position of the cells within the cycle at the time of cooling to 4°C the data shown in Figure 2(b) and (c) were obtained. In Figure 2(b) a very late S-phase cell cycle marker was used. Because it is well known that Chinese hamster cell lines have a very resistant period to X-ray radiation inactivation at the end of the DNA synthesis period, cells were irradiated at various times after release from the hydroxyurea block with 1150 rad of X-rays (145 kV; 153 rad/ min) (Burki & Carrsno, 1973). The data indicate that the majority of cells in this
CRITICAL M
G,,, +
IO v) = s 0
8-t
I
L)Nd
hydroxy urea block I
SI I
I 4; I I I 2 -1
a,
A ABlock --.-..-----•
I O-’ @~--‘-----l~----~ 2 4 0 Time afte: mltotic
SI
T : : s -0 g, : P
.I I
1 (a) I
6-1
5 kl a
6
release -1 IO
8
cell detachment
a
(h)
%,I
Time of cooling
(b)
- 100
1
I I
z E 6 0) $
IO=
603
DAMAGE
/“\,’ 0
I60 \ ?,
0.1 i
5
i 0 “Lo
001. 0
1 I 2
I 4
Time ofter hydroxyureo removal (h)
I 6
B
0
2
4
6
8
I
Time after release from hydroxyureo (h)
FIG. 2. (a) The percentage of mitotic cells (a), and the percentage of the synchronized cells incorporating high specific activity tritiated thymidine (A), is a function of time after initial selection of mitotic cells which are then grown in a media containing hydroxyurea for 7 h and then washed and grown in fresh media at 37’C. (b) The percentage survival of the synchronized cell population exposed to 1150 rads of roentgen radiation at different times after the initial hydroxyurea block is removed. (c) The number of colonies per 1000 cells after a second exposure of hydroxyurea, as a function of time after release from the first hydroxyurea block for different experimental formats. (A) No IdUrd; (0) 10-s M-IdUrd and washing; (;?) 10-s M-IdUrd fir&half S period; (0) 10-s M-TdUrd second-half of the S period.
synchronous population reached their resistant stage at five hours. The data given in Figure 2(c) can be used to indicate the time of entrance into the G, period as a result of the different experimental formats. The different experimental procedures did not effect the time of entrance into the G, period. After hamster cells were placed in the refrigerator the progress through the cell cycle rapidly slowed down and macromolecular synthesis was reduced to nearly zero (Nelson et al., 1971). During four days storage at 4°C the cells progressed slowly toward the M stage (Burki, unpublished observations).
H.
604
(c) Killing
eficiency
J.
BURKI
of [‘251]DNA disintegrations
in different
intranuclear
regions
The results of a typical experiment are given in Figure 3. The slope of the survival curve was zero for controls and for IdUrd alone, and increased as the t,ota,l activity of the 1251 in the medium increased. However? the highest total activity experiments showed a resistant component on the survival curve at longer times. This resistant component was always seen and varied with the highest amount of lz51 used in the experiments. When data of the type given in Figure 2 were related to the total number of 1251 decays in DNA, the results in Figure 4 were found. Because each experiment was a composite of three dose rates, and the combined data fell on a straight line (Fig. 4), dose rate effects are not reflected in these experiments. The survival curves shown in Figures 3 and 4 are at least biphasic, including the curves for cells labeled for the
0
10
20
30
40
50
60
70
Time of storage at 4°C in growth media (h)
FIG. 3. The measured percentage survival ment at 4 different levels. (0) Just 10e6 [‘26I]dUrd (0) as a function of the number
for cells incorporating iwIdUrd; and either of hours at 4°C.
in a typical experi[ 1251]dUrd low, medium, or high levels of
complete DNA synthesis period (Fig. 4(b)). The initial slope of the survival curve for Figure 4(a) (first half of the S period) was O-031, corresponding to an initial Do7 of 32.3 decays, whereas the cells labeled for the complete S period had an initial slope of 0.042, corresponding to a D,, of 23.8 decays. The steepest initial slope is given in Figure 4(c) for the cells that incorporated [ 1251]dUrd in the second half of the DNA synthesis period, which has a slope of 0.063 or a D,, of 15.9 decays. In a preliminary study (Burki, 1974) the initial slope difference between [ 1251]dUrd incorporated during early S and late S period was measured for small amounts of [1251]dUrd. In that study there was still a possibility that resistant components of the complex survival curve might influence the observed differences in the efficiencies of inactivation. For this reason, experiments were conducted with much higher amounts of [1251]dUrd to the number of decays to reduce the survival by l/e or e-l t Abbreviations used : D,, (e = 2.7182) in the linear portion of the survival curve (used when the survival curve has a of decays to kill 63% of the cells in a simple exponential survival shoulder); D,,, the numljer curve.
CRITICAL
DNS
005
DAMAGE
I 200
I 100
0
100
200 No. of ‘*’
300 I decays
400
600
000
3c
IOC
m ONA
4. (a) Survival curve for synchronized cells stored at 4’C in the Gz period after incorporating [lz51]dUrd into the DNA replicated in the first half of the S period. Composite of several individual experiments (0); individual experimental results ( 0). Error bars are either standard error of the mean or standard deviations. (b) Survival curves for synchronized cells stored at 4°C in the Gz period after incorporating [‘251]dUrd unifilarily into the total replicated DNS. Results from two experiments. Symbols as in (a). (c) Survival curves for synchronized cells stored at 4°C’ in the GZ period after incorporating [‘251]dUrd into the DNA replicated in the second half of the S period. Composite results of 6 experiments (0). Two other series of experiments at higher dose levels (0) and ( 3). FIG.
investigate the more resistant components of the survival curves. The results show that there were resistant components on all of the observed survival curves, including curves for cells that had been labeled for one complete round of DNA replication (Fig. 4). These resistant components permit a resolution of the survival curves into at, least two components in all three cases shown in Figure 4. For the first half of the S period the resolved initial component was 30 decays; for the unifilar labeling the initial component was 15 decays ; and for the second half of the S period the initial component
606
H.
J.
BURKl
was 11 decays. The important point to note is that the differences continued to exist even when these complex survival curves were resolved. However, for simplicity, we elected to take the uncorrected initial slope of the survival curves. The survival curve “resistant, components” have another important significance in these studies. They could be due either to a small fraction of the population of cells that are extremely resistant to [ lz51 ] dUrd decays in cell DNA, or to heterogeneous distribution of label among cells. These two possibilities were distinguished by autoradiography. If there were slow growing cells in t’he populat’ion, or cells which incorporated less [ 1251]dUrd than other cells, the histograms of the grain distribution should be quite widespread. If there was a small variation in the grains per cell the survival curve “tails” would be due to extremely resistant cells. Figure 5(a) shows the histogram of the grain distribution for cells labeled in the first half of the S period. while Figure 5(b) shows the data for t.he second half. In both cases the distribution of grains is rather large and there is a fraction of cells which is lightly labeled. This fraction explains the resistant component of the survival curves. The lightly labeled fractions seen in Figure 5(a) and (b) were similar. In autoradiographs of cells that’ were exposed for longer times there always existed a Sq/, or smaller fraction of very lightly labeled cells. The reason for this heterogeneous labeling is not clear. WC also noted that the film efficiency found in the first half of the S period \vas I 1’:; compared to 14% in the second half, a difference which probably reflect’s the peripheral distribution of DNA replication in hamster cells in the second half of the S period (Huberman et al., 1973 ; Comings & Okada, 1973). After confirming that the resistant component of the survival curve was due to lightly labeled cells in all the labeling procedures, 25 experiments were performed with various experimental formats to determine the efficiency of inactivation of cells for
Second half of S period
FIG? half of S permd
241
0
10 20 30 40 50 60 70 No. of silver (a)
grains (b)
FIQ. 5. Histogram of the number of silver grains per cell after the synchronous population had incorporated [lasI]dUrd into the DNA synthesis period in the first half of the DNA synthesis period (a); and the second half of the DNA synthesis period (b).
CRITICAL
DNA
DAMAGE
ties
Midtime of labeling period in S period (h) Fm. 6. The initial cell inactivation efficienoyt per izsI disintegration into time of the labeling period in which [ ‘a51]dUrd was incorporated labeled from 3 to 6 h in the second half of the DNA synthesis period the t Delined as 1/iD3, = i.e., where ,D,, equals the dose to kill 63% portion of the cell survival curve.
as a function of the midcell DNA (e.g., for oells midtime is at 4.6 h). of t,he cells in the initial
damage accumulated in various DNA regions. A summary of the results is given in Figure 6. These results show that the killing efficiency of [lz51]DNA depended on the site of the lz51 decays within the DNA. The initial D,, was 62.5 decays for the DNA labeled in the first quarter of the DNA synthesis period, 40 for the first third, 32 for the first half, 28 for the third quarter, 16 for the second half or two-thirds and 18 for DNA synthesized in the last quarter. The killing efficiency appeared to be at a maximum between the fourth and the fifth hour of the S period, although the results for the last quarter of the S period were not statistically different from those at maximum efficiency.
4. Discussion (a) The critical
DNA
damage hypothesis
The initial cell inactivation efficiency per lZ51 decay changed depending on what part of the DNA was labeled and was damaged by the decay of this isotope. In those experiments in which Chinese hamster cells were stored at 4°C at the G, + M stage of the cell cycle there appears to be a maximum sensitivity for cells which receive damage in the DNA which replicates about three-quarters of the way through the S period. This damage to late replicating DNA is critical in preventing the reproduction of the Chinese hamster cell. The most likely reason why this damage is critical is because it leads to lethal chromosome aberrations. The recent finding that [1251]dUrd decays in DNA are efficient inducers of dicentric and ring chromosomes, with 35 lz51 decays leading to one dicentric or ring chromosome per cell on the average, is important in this regard (Burki et al.. 1976). Since this dose of 35 lz51 decavs , is similar t’o the D,, for hamster
608
H.
J.
BURKI
cells, this suggests that the lethal event caused by lz51 decay is the induction of dicentrics or ring chromosomes. Recently, Hughes et al. (1976) have shown that lz51 decays induce chromatid aberrations in G, + M cells at the sites where these decays occur. Thus, our hypothesis is that regions exist in mammalian cell DNA where 125I damage leads to chromosome aberrations and to reproductive death more efficiently than in other regions, and that these critical regions are located in the DNA that replicates three-quarters of the way through the S period in Chinese hamster cells. There are many data to support the hypothesis that chromosome aberrations induced by ionizing radiation are non-random in many different systems, including Vicia $&a (Revell, 1953), rat kangaroo (Van Stennis et al., 1974), and human cells (Seabright, 1973; Caspersson et al., 1972). There appears to be evidence that the heterochromatic regions of the chromosomes are the regions where chromosome aberrations occur in these systems. Because most of the heterochromatic parts of chromosomes contain DNA that replicates late, the results reported here confirm these earlier observations. Another way to explain that 125I decays lead to reproductive death with different efficiencies in different parts of the cell DNA, is to propose that some regions of the interphase chromatin are less accessible to repair of 1251-induced damage than other regions. This hypothesis would predict that the reason for the differences depicted in Figure 6 is that the DNA that replicates late in the DNA synthesis period is less accessible to repair and may contain a higher percentage of non-repairable damage. This repair hypothesis is not incompatible with the critical DNA damage hypothesis proposed above because critical chromosome damage still could be the reason for cell inactivation, although it could result from lack of repair. (b) Characteristics
of lute replicating
DNA irb mammalian
cells
Generally the hamster DNA that replicates late in the DNA synthesis period is heterochromatic (Comings, 1972b), although there is still the possibility that certain critical messengers necessary for mitosis or some other vital function also replicate late. It is known that parts of chromosomes replicate at different times in the S phase. In the mouse, for example, centromeric heterochromatin replicates in the third quarter of the S period (Bostock & Prescott, 1971b). In the hamster, centromeric heterochromatin is replicated at approximately four to five hours before mitosis of chromosomes 4 and 5, but certain other chromosomes replicate their centromeres at a later time (Hsu, 1964). Recently, Stubblefield (1975) has shown that most of the regions near the centromeres of hamster chromosomes contain DNA that replicates in the last third of the DNA synthesis period. We can infer from the results of Stubblefield (1975) and others that our proposed critical damage region is associated with the regions of DNA that form the centromeric and near-centromeric regions of chromosomes. Much of the late-replicating DNA replicates in the periphery of the nucleus, near the nuclear membrane (Huberman et al., 1973 ; Comings & Okada, 1973). The fact that this region within the nucleus may be critical has also been suggested by electron penetration experiments (Zermeno & Cole, 1969), where a thin shell critical region including the nuclear membrane has been proposed. This region must contain late replicating centromeric or near-centromeric DNA
CRITICAL
DNA
(c) Other consequences of the critical
609
DAMAGE
DNA
damage hypothesis
One of the striking relationships between the killing efficiency and time of labeling (Wig. 6) is the similarity of the shape of the curve to the shape of the survival curve for Chinese hamster cells exposed to a fixed dose of ionizing radiation during the S period (Bird & Burki, 1975). The two sets of data, although from completely different types of experiments, might be related if one assumes that for replication the critical DNA must be made more diffuse than its normal condensed state. Thus the maximum resistance to ionizing radiation that occurs just before the end of the S period may be related to the fact that critical near-centromeric and centromeric DNA is diffuse and heing replicated at that point. With low linear energy transfer radiation such as S-rays or tritium, much of whose damage might be repaired if there was access to the site before chromosome aberrations are formed, this critical DNA would have the greatest possibility of being repaired during the part of the cell cycle when it was most diffuse. This is similar to the hypothesis of Dewey et al. (1972) formed from their observation that the differential sensitivity to radiation during the cell cycle is greatly diminished by exposure of cells to radiation in a state where all the chromatin was forced to remain more condensed. They found that cell-cycle differences in radiation sensitivity were reduced by hypertonic treatment. The most pronounced effect was to sensitize cells of the late S period. Another consequence of the possible existence of critical DNA damage regions in mammalian cells is that these critical regions might be replicated at different times in t>he S period for different species, and they might be a larger or smaller fraction of the total cell DNA for different species. For example, if mouse satellite DNA was whcrc> critical DNA damage occurred, this might explain the greater radiosensitivity of some neer-diploid cells such as L5178Y cells (Burki et al., 1973), and might also partially explain why t.he response t’o ionizing radiation is different in the mouse during t’he cell a?-cle and whp it is not the same as seen in human and hamster cells. TIw competent technical assistance of Mrs Catherine Lathrop in this work is greatly appreciated. Manuscript preparation was by MS Mary Graham. This investigation was supported by the National Institutes of Health (grant CA-14310) and a grant from the ITT.S. b:rlcr~~
Research
and Development
Administration.
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