- Email: [email protected]
Cell stages refractory to thymidine incorporation induced by X-rays
Experimental
Cell Research
37, 39-44
39
(1965)
CELL STAGES REFRACTORY TO THYMIDINE INCORPORATION INDUCED BY X-RAYS R. A. McGRATH, Biology Division, and Department
W.
M.
LEACH
and J.
G. CARLSON
Oak Ridge National Laboratory,l Oak Ridge, of Zoology and Entomology, The University Tennessee,’ Knoxville, Term., U.S.A. Received
February
Term., of
14, 1964
the basis of 14C or sH thymidine incorporation, it is known that synthesis of deoxyribonucleic acid (DNA) in grasshopper neuroblasts begins in middle telophase and ends in very early prophase [4, 71. When neuroblasts are exposed to 32 or 250 r of X-rays after normal synthesis of DNA (S) has ended, they incorporate additional 3H-thymidine (3HTdR) into chromosomal DNA [7]. Incorporation is greater in early prophase (near the end of S) than in later prophase stages. The purpose of the present study was to determine whether neuroblasts in prometaphase, metaphase, anaphase, and early telophase could incorporate 3HTdR after exposure to X-rays. ON
MATERIALS
AND
METHODS
Embryos of the grasshopper Chortophaga viridifasciata (De Geer) at an age equivalent to 14 days of development at 26°C were used. Eggs were placed in 70 per cent ethanol for about 1 min, dried on sterile filter paper, and then transferred to Shaw’s [9] culture medium. Embryos were removed from eggs and separated from surrounding membranes and yolk. Tissues lateral to the nerve cord were cut away, and the remaining strip of tissue (from the last maxillary through first abdominal segments) was transferred to culture medium which contained 3HTdR3 alone or 1 x 1O-6 M colchicine4 plus 3HTdR. Hanging-drop preparations were made [2] and examined microscopically. A constant-temperature incubator which enclosed the lower part of the microscope was maintained at 38 jIO.5”C. Several neuroblasts in each preparation were mapped [2, 71 to ensure their reidentification at later times. Thirty min after the start of treatment with colchicine and/or 3HTdR, preparations were exposed to X rays. Both a 250- and a 300-KV G.E. Maxitron was used under the follow1 Operated by Union Carbide Corporation for the US Atomic Energy Commission. e Supported in part by US Atomic Energy Commission grant No. AT-(40-l)-2575. 3 Obtained from New England Nuclear Co. and checked chromatographically. Samples with greater than 2 per cent 3H contamination were not used. Final specific activity of material used in this study was 0.5 C/mM. 4 At this concentration, colchicine blocks mitotic progress of grasshopper neuroblasts by preventing spindle formation [5]; blocked cells are referred to as “C-mitotic” or “C-metaphases.” Experimental
Cell
Research
37
40
R. A. XcCrath,
IV. AI. Leach and J. G. Carlson
ing conditions: IO mA, 100 KVP, target to object distance of 10 or 20 cm, 0.28- or 0.5-mm Al filter, and exposure rates in air of approximately 400 r/min. After exposure, previously mapped neuroblasts were relocated. Thirty to 60 min after the end of X-ray treatment, preparations were fixed in 50 per cent acetic acid, embedded in paraffin, and sectioned at 6 p. Autoradiograms were prepared and previously mapped neuroblasts were relocated. In some cases squash preparations were made from hanging-drop preparations [S]. Most of the methods used for this study were similar to those previously described [2, 7, 81. Additional technical details are given, where relevant, in the text. RESULTS
AND
CONCLUSIONS
The use of colchicine to prevent cells from entering anaphase and of irradiation to stop cell progress before prometaphasel is shown in the following experiments. Thus cells that had passed through the critical stage of radiation sensitivity (see Fig. 1) were trapped in C-mitosis. Fig. 2 depicts the untreated condition. The average number of mitotic neuroblasts within the three thoracic segments of the embryo (about SO neuroblasts/segment) is plotted against time in Shaw’s solution containing 10 PC/ml of 3HTdR. The number of cells at each mitotic stage (counted at 10 min intervals) did not change during the first 90 min after hanging-drop preparations were made. Fig. 3 shows the average cell number plotted against time in Sham’s medium which contained 10 PC/ml 3HTdR plus 1 X 10-G M colchicine. The number of anaphases plus early telophases decreased to 0 within 60 min. During the same time the number of prometaphases plus metaphases and C-metaphases increased by a factor of about 5. The rate of accumulation CRITICAL
STAGE
COLCHICINE
&
A
& (’
BLOCK
INT
VEP
EP
-ONE
MPLP,zI;:MT :
LT
INT VEP
EP
1
i- 60 min-1 CvCLE=208min-
Fig. l.-Important times in the grasshopper-neuroblast cell cycle. I, period of X-ray-induced incorporation of 3HTdR; S, period of DNA synthesis. P, time at which pool of TdR derivatives was formed. INT, interphase; VEP, very early prophase; EP, early prophase; MP, middle prophase; LP, late prophase; VLP, very late prophase; PM, prometaphase; M, metaphase; ANA, anaphase; ET, early telophase; MT, middle telophase; LT, late telophase. 60 min arrow indicates elapsed time from the start of middle telophase. Total cell cycle time at 38”C, 208 min. Time of cell stages from Carlson [3]. 1 Neuroblasts pass through a critical stage of radiation down of the nuclear boundary. Cells exposed to X-rays to earlier mitotic stages, whereas cells that have passed Experimental
Cell Research
37
sensitivity about 5 min before breakbefore this stage are delayed or revert this stage continue through mitosis [l].
41
Cell stage dependence
within the latter population was approximately 3 cells/l0 min under these experimental conditions. Fig. 4 depicts the colchicine-S-ray cell trap. The average number of mitotic figures is plotted against time in Shaw’s medium which contained 10 ,w/ml 3HTdR plus 1 X IO-6 M colchicine as above. Preparations were exposed to 64 r of X-rays 30 min after the start oftreatment.
”
LIt
30 60 TiME lrni”
Fig.
2.
90
30 60 TIME (ml*)
Fig.
3.
Fig. 2.-Relationship between number of mid-mitotic neuroblasts and time in culture medium plus 10 ,uc/ml 3HTdR. 0, Anaphase plus early telophase; 0, prometaphase plus metaphase. Bars indicate 95 per cent confidence levels. Each point represents an average of nine observations. Fig. plus
3.-Relationship 10 PC/ml 3HTdR
between number of mid-mitotic neuroblasts and time in culture and 1 x lO-6 M colchicine. Symbols 0, o same as in Fig. 2.
medium
The initial increase in the C-mitotic population was about 3 cells per 10 min for approximately 50 min. No further increase occurred between 50 and 150 min because prior to the critical stage, cells exposed to X-rays are delayed mitotically or revert to earlier mitotic stages. Neuroblasts in the population after 50 min (2-4 times the normal prometaphase-metaphase cell number) were those trapped by colchicine treatment. Autoradiograms of mid-mitotic neuroblasts treated with colchicine plus 3HTdR or with colchicine plus 3HTdR and X-rays were not labeled above background levels. When colchicine-treated cells in the star configuration1 were exposed to 250 or 5000 r of X-rays, the chromosomes fused into an irregular vesiculated mass. Vesiculation was seen whether or not the culture medium contained 3HTdR. Neither fusion nor vesiculation was seen when only the medium 1 Thirty ends come
to 40 min after start of treatment with 1 x 10-e A4 colchicine the proximal together to form a three dimensional “star.” This orientation lasts for Experimental
chromosome several hr.
Cell
Research
37
42
R. 4. McGrath,
W. M. Leach and J. G. Carlson
(with colchicine and/or 3HTdR) was exposed to 5000 r of X-rays. Fusion of star metaphases and vesicle formation within the fused mass is therefore an effect of X-rays on the cell and not a secondary effect on the medium. Autoradiographic analysis of squash preparations of fused, vesiculated neuroblast chromosomes showed no incorporation of 3HTdR after X-ray exposures of 250, 5000, or 75,000 r (the last at an exposure rate of 4000 r/min). 25 20
0 0-h-u-.-.-.->?30 60 TIMEcm,ni Fig.
ov, I,, I 0 10 20 30 40 50 60 70 TIME(ml”)
90 150
4.
Fig.
5.
Fig. 4.-Relationship between number of mid-mitotic neuroblasts and time in culture medium plus 10 pc/ml 3HTdR and 1 x 1O-B M colchicine. Preparations exposed to 64 r of X-rays at 30 min. l , symbols o, same as in Fig. 2. Fig. text
5.-Grain for details.
number
per unit
area
plotted
against
time
from
start
of middle
telophase.
See
Cells in the period between anaphase and the start of DNA synthesis were studied in the following experiment. Preparations were made up in medium containing 10 PC/ml 3HTdR but no colchicine. Metaphase neuroblasts were exposed to 1000 r of X rays and preparations were fixed at early telophase, before the beginning of normal DNA synthesis (see Fig. 1). No labeling was observed. These colchicine studies show that neuroblasts between the critical stage of radiation sensitivity in prophase and the start of DNA synthesis (midtelophase) are not stimulated to incorporate 3HTdR by X radiation. 3HTdR which enters neuroblasts in prophase is carried through midmitosis presumably as a “pool” of thymidine derivatives, and incorporated into DNA during the following S period [6]. The following experiment was designed to test whether changes in the nucleotide pool could account for failure of radiation to stimulate TdR incorporation during mid-mitosis. Experimental
Cell Research
37
Cell stage dependence
43
Embryos were placed in culture medium which contained 10 PC/ml 3HTdR for 10 min and then rinsed in two changes (5 min each) of culture medium containing unlabeled thymidine 100 times more concentrated than the 3HTdR. Embryos were then rinsed in isotonic culture medium and made into hangingdrop preparations. Mid-mitotic neuroblasts were mapped for later reidentification. When the mapped cells were 60 min past the start of middle telophase (see Fig. l), preparations were fixed, autoradiograms were prepared, and grain number per unit area was counted over the previously mapped cells (Fig. 5). These grains must have resulted from a pool formed during midmitosis. The values plotted between 30 and 40 min in Fig. 5 were obtained from experiments in which embryos were exposed to 32 r of X-rays immediately before or after the change from 3HTdR into nonlabeled TdR. A straight line from the mean of the 60-min grain numbers through the origin passes through the mean of the 30-40 min values.1 These results indicate that exposure of neuroblasts to X-rays during formation of a pool of thymidine derivatives had no detectable effects on incorporation of 3H into DNA during the first 30 min of the following S period. Within the limits of the experimental conditions, failure to induce incorporation of 3HTdR derivatives into DNA by exposure of mid-mitotic neuroblasts to X-rays was not due to absence of or damage to a TdR-derived pool. DISCUSSION
These results show that incorporation of 3HTdR derivatives into chromosomal DNA cannot be induced by X irradiation of grasshopper neuroblasts between the critical stage in very late prophase and the beginning of middle telophase (when normal DNA synthesis starts). Within the experimental limits of this study, the effects of irradiation on formation and utilization of a pool of thymidine derivatives are negligible. Failure of a precursor to enter and be retained within the cell, therefore, is probably not the factor responsible for absence of radiation-induced incorporation. We assume that a particular series of events or set of enzymatic conditions constitute a DNA synthesis-like (S-like) environment within the cell and that normal synthesis of DNA begins when the S-like environment is established and continues until replication is complete. The data presented in this paper and a previous paper [7] fit the following concept. 1 The average grain number (23.4) start of telophase until preparations an X-ray exposure of 512 r. Average tion = 16-18; 512 r = 13-16.
has been normalized to 35.4 min, the average time from the were fixed. The experiment described was repeated with grain numbers per 14.6 p2 at 30 to 40 min were: no irradia-
Experimental
Cell Research
37
44
R. A. McGrath,
W. M. Leach and J. G. Carlson
When replication of DNA is finished, the S-like environment does not immediately disappear but is gradually reduced as neuroblasts progress beyond the S period. The period of reduction corresponds to the time during which mitotic delay or reversion can be induced by irradiation (from very early prophase until the critical stage) and also to the stages of S-ray-induced incorporation of 3HTdR derivatives into chromosomal DNA. (This may also be the period when repair of radiation damage occurs.) Once the cells progress to the critical stage in very late prophase, the S-like condition is reduced to such a level that X-ray-induced incorporation of 3HTdR cannot occur. No mitotic delay or reversion occurs after this stage. Whatever damage has been done remains, and may or may not be corrected at some later cell time. It is obvious that Y-ray-induced incorporation of 3HTdR derivatives into chromosomal DNA is related to intracellular environmental conditions. Our data imply in addition a correlation to cell stage at the time of radiation exposure. SUMMARY
Autoradiographic studies show that grasshopper neuroblasts, trapped in mid-mitotic stages by colchicine and then exposed to 64 to 75,000 r of X-rays do not incorporate 3HTdR or its derivatives into DNA. The intracellular pool of TdR derivatives is not detectably changed by exposure to S-rays. The data show a correlation between radiation-induced incorporation of 3HTdR derivatives and cell stage at the time of irradiation. It is suggested that the synthesis-like condition, during which S-ray-induced incorporation of DN;A precursors can occur, degenerates gradually after cells stop normal synthesis of DNA. The comments and criticisms of Drs R. B. Setlow, R. F. Kimball and D. M. Prescott and discussions with them of various aspects of this work are gratefully acknowledged. Also we thank Dr S. Wolff for providing the colchicine used in this study. REFERENCES 1. CARLSON, J. G., Cold Spring Harbor Symp. 9, 104 (1941). 2. CARLSOX, J. G. and GAULDEN, M. E., in D. M. PRESCOTT (ed.) Methods in Cell Physiology, vol: 1. Academic Press, New York, 1964. 3. CARLSON, J. G. and HOLLAENDER, A., J. Cellular Comp. Physiol. 31, 149 (1948). 4. GAULDEN, M. E., Genetics 41, 645 (1956). 5. GAULDEN, M. E. and CARLSON, J. G., Expf! Cell Res. 2, 416 (1951). 6. LEACH. W. M.. Am. Sot. Biol. Bull. 10, 32 (1963). 7. MCGR~TH, R. A., Radiation Res. 19, 526 (1663). ’ 8. MCGRATH. R. A.. LEACH. W. M. and CARLSON, J. G., J. Roy. Microscop. Sot. 82, 55 (1963). 9. SHAW, E’I., Ex&Z Cell Res. 11, 580 (1956)
Experimental
Cell Research
37
Cell Research
37, 39-44
39
(1965)
CELL STAGES REFRACTORY TO THYMIDINE INCORPORATION INDUCED BY X-RAYS R. A. McGRATH, Biology Division, and Department
W.
M.
LEACH
and J.
G. CARLSON
Oak Ridge National Laboratory,l Oak Ridge, of Zoology and Entomology, The University Tennessee,’ Knoxville, Term., U.S.A. Received
February
Term., of
14, 1964
the basis of 14C or sH thymidine incorporation, it is known that synthesis of deoxyribonucleic acid (DNA) in grasshopper neuroblasts begins in middle telophase and ends in very early prophase [4, 71. When neuroblasts are exposed to 32 or 250 r of X-rays after normal synthesis of DNA (S) has ended, they incorporate additional 3H-thymidine (3HTdR) into chromosomal DNA [7]. Incorporation is greater in early prophase (near the end of S) than in later prophase stages. The purpose of the present study was to determine whether neuroblasts in prometaphase, metaphase, anaphase, and early telophase could incorporate 3HTdR after exposure to X-rays. ON
MATERIALS
AND
METHODS
Embryos of the grasshopper Chortophaga viridifasciata (De Geer) at an age equivalent to 14 days of development at 26°C were used. Eggs were placed in 70 per cent ethanol for about 1 min, dried on sterile filter paper, and then transferred to Shaw’s [9] culture medium. Embryos were removed from eggs and separated from surrounding membranes and yolk. Tissues lateral to the nerve cord were cut away, and the remaining strip of tissue (from the last maxillary through first abdominal segments) was transferred to culture medium which contained 3HTdR3 alone or 1 x 1O-6 M colchicine4 plus 3HTdR. Hanging-drop preparations were made [2] and examined microscopically. A constant-temperature incubator which enclosed the lower part of the microscope was maintained at 38 jIO.5”C. Several neuroblasts in each preparation were mapped [2, 71 to ensure their reidentification at later times. Thirty min after the start of treatment with colchicine and/or 3HTdR, preparations were exposed to X rays. Both a 250- and a 300-KV G.E. Maxitron was used under the follow1 Operated by Union Carbide Corporation for the US Atomic Energy Commission. e Supported in part by US Atomic Energy Commission grant No. AT-(40-l)-2575. 3 Obtained from New England Nuclear Co. and checked chromatographically. Samples with greater than 2 per cent 3H contamination were not used. Final specific activity of material used in this study was 0.5 C/mM. 4 At this concentration, colchicine blocks mitotic progress of grasshopper neuroblasts by preventing spindle formation [5]; blocked cells are referred to as “C-mitotic” or “C-metaphases.” Experimental
Cell
Research
37
40
R. A. XcCrath,
IV. AI. Leach and J. G. Carlson
ing conditions: IO mA, 100 KVP, target to object distance of 10 or 20 cm, 0.28- or 0.5-mm Al filter, and exposure rates in air of approximately 400 r/min. After exposure, previously mapped neuroblasts were relocated. Thirty to 60 min after the end of X-ray treatment, preparations were fixed in 50 per cent acetic acid, embedded in paraffin, and sectioned at 6 p. Autoradiograms were prepared and previously mapped neuroblasts were relocated. In some cases squash preparations were made from hanging-drop preparations [S]. Most of the methods used for this study were similar to those previously described [2, 7, 81. Additional technical details are given, where relevant, in the text. RESULTS
AND
CONCLUSIONS
The use of colchicine to prevent cells from entering anaphase and of irradiation to stop cell progress before prometaphasel is shown in the following experiments. Thus cells that had passed through the critical stage of radiation sensitivity (see Fig. 1) were trapped in C-mitosis. Fig. 2 depicts the untreated condition. The average number of mitotic neuroblasts within the three thoracic segments of the embryo (about SO neuroblasts/segment) is plotted against time in Shaw’s solution containing 10 PC/ml of 3HTdR. The number of cells at each mitotic stage (counted at 10 min intervals) did not change during the first 90 min after hanging-drop preparations were made. Fig. 3 shows the average cell number plotted against time in Sham’s medium which contained 10 PC/ml 3HTdR plus 1 X 10-G M colchicine. The number of anaphases plus early telophases decreased to 0 within 60 min. During the same time the number of prometaphases plus metaphases and C-metaphases increased by a factor of about 5. The rate of accumulation CRITICAL
STAGE
COLCHICINE
&
A
& (’
BLOCK
INT
VEP
EP
-ONE
MPLP,zI;:MT :
LT
INT VEP
EP
1
i- 60 min-1 CvCLE=208min-
Fig. l.-Important times in the grasshopper-neuroblast cell cycle. I, period of X-ray-induced incorporation of 3HTdR; S, period of DNA synthesis. P, time at which pool of TdR derivatives was formed. INT, interphase; VEP, very early prophase; EP, early prophase; MP, middle prophase; LP, late prophase; VLP, very late prophase; PM, prometaphase; M, metaphase; ANA, anaphase; ET, early telophase; MT, middle telophase; LT, late telophase. 60 min arrow indicates elapsed time from the start of middle telophase. Total cell cycle time at 38”C, 208 min. Time of cell stages from Carlson [3]. 1 Neuroblasts pass through a critical stage of radiation down of the nuclear boundary. Cells exposed to X-rays to earlier mitotic stages, whereas cells that have passed Experimental
Cell Research
37
sensitivity about 5 min before breakbefore this stage are delayed or revert this stage continue through mitosis [l].
41
Cell stage dependence
within the latter population was approximately 3 cells/l0 min under these experimental conditions. Fig. 4 depicts the colchicine-S-ray cell trap. The average number of mitotic figures is plotted against time in Shaw’s medium which contained 10 ,w/ml 3HTdR plus 1 X IO-6 M colchicine as above. Preparations were exposed to 64 r of X-rays 30 min after the start oftreatment.
”
LIt
30 60 TiME lrni”
Fig.
2.
90
30 60 TIME (ml*)
Fig.
3.
Fig. 2.-Relationship between number of mid-mitotic neuroblasts and time in culture medium plus 10 ,uc/ml 3HTdR. 0, Anaphase plus early telophase; 0, prometaphase plus metaphase. Bars indicate 95 per cent confidence levels. Each point represents an average of nine observations. Fig. plus
3.-Relationship 10 PC/ml 3HTdR
between number of mid-mitotic neuroblasts and time in culture and 1 x lO-6 M colchicine. Symbols 0, o same as in Fig. 2.
medium
The initial increase in the C-mitotic population was about 3 cells per 10 min for approximately 50 min. No further increase occurred between 50 and 150 min because prior to the critical stage, cells exposed to X-rays are delayed mitotically or revert to earlier mitotic stages. Neuroblasts in the population after 50 min (2-4 times the normal prometaphase-metaphase cell number) were those trapped by colchicine treatment. Autoradiograms of mid-mitotic neuroblasts treated with colchicine plus 3HTdR or with colchicine plus 3HTdR and X-rays were not labeled above background levels. When colchicine-treated cells in the star configuration1 were exposed to 250 or 5000 r of X-rays, the chromosomes fused into an irregular vesiculated mass. Vesiculation was seen whether or not the culture medium contained 3HTdR. Neither fusion nor vesiculation was seen when only the medium 1 Thirty ends come
to 40 min after start of treatment with 1 x 10-e A4 colchicine the proximal together to form a three dimensional “star.” This orientation lasts for Experimental
chromosome several hr.
Cell
Research
37
42
R. 4. McGrath,
W. M. Leach and J. G. Carlson
(with colchicine and/or 3HTdR) was exposed to 5000 r of X-rays. Fusion of star metaphases and vesicle formation within the fused mass is therefore an effect of X-rays on the cell and not a secondary effect on the medium. Autoradiographic analysis of squash preparations of fused, vesiculated neuroblast chromosomes showed no incorporation of 3HTdR after X-ray exposures of 250, 5000, or 75,000 r (the last at an exposure rate of 4000 r/min). 25 20
0 0-h-u-.-.-.->?30 60 TIMEcm,ni Fig.
ov, I,, I 0 10 20 30 40 50 60 70 TIME(ml”)
90 150
4.
Fig.
5.
Fig. 4.-Relationship between number of mid-mitotic neuroblasts and time in culture medium plus 10 pc/ml 3HTdR and 1 x 1O-B M colchicine. Preparations exposed to 64 r of X-rays at 30 min. l , symbols o, same as in Fig. 2. Fig. text
5.-Grain for details.
number
per unit
area
plotted
against
time
from
start
of middle
telophase.
See
Cells in the period between anaphase and the start of DNA synthesis were studied in the following experiment. Preparations were made up in medium containing 10 PC/ml 3HTdR but no colchicine. Metaphase neuroblasts were exposed to 1000 r of X rays and preparations were fixed at early telophase, before the beginning of normal DNA synthesis (see Fig. 1). No labeling was observed. These colchicine studies show that neuroblasts between the critical stage of radiation sensitivity in prophase and the start of DNA synthesis (midtelophase) are not stimulated to incorporate 3HTdR by X radiation. 3HTdR which enters neuroblasts in prophase is carried through midmitosis presumably as a “pool” of thymidine derivatives, and incorporated into DNA during the following S period [6]. The following experiment was designed to test whether changes in the nucleotide pool could account for failure of radiation to stimulate TdR incorporation during mid-mitosis. Experimental
Cell Research
37
Cell stage dependence
43
Embryos were placed in culture medium which contained 10 PC/ml 3HTdR for 10 min and then rinsed in two changes (5 min each) of culture medium containing unlabeled thymidine 100 times more concentrated than the 3HTdR. Embryos were then rinsed in isotonic culture medium and made into hangingdrop preparations. Mid-mitotic neuroblasts were mapped for later reidentification. When the mapped cells were 60 min past the start of middle telophase (see Fig. l), preparations were fixed, autoradiograms were prepared, and grain number per unit area was counted over the previously mapped cells (Fig. 5). These grains must have resulted from a pool formed during midmitosis. The values plotted between 30 and 40 min in Fig. 5 were obtained from experiments in which embryos were exposed to 32 r of X-rays immediately before or after the change from 3HTdR into nonlabeled TdR. A straight line from the mean of the 60-min grain numbers through the origin passes through the mean of the 30-40 min values.1 These results indicate that exposure of neuroblasts to X-rays during formation of a pool of thymidine derivatives had no detectable effects on incorporation of 3H into DNA during the first 30 min of the following S period. Within the limits of the experimental conditions, failure to induce incorporation of 3HTdR derivatives into DNA by exposure of mid-mitotic neuroblasts to X-rays was not due to absence of or damage to a TdR-derived pool. DISCUSSION
These results show that incorporation of 3HTdR derivatives into chromosomal DNA cannot be induced by X irradiation of grasshopper neuroblasts between the critical stage in very late prophase and the beginning of middle telophase (when normal DNA synthesis starts). Within the experimental limits of this study, the effects of irradiation on formation and utilization of a pool of thymidine derivatives are negligible. Failure of a precursor to enter and be retained within the cell, therefore, is probably not the factor responsible for absence of radiation-induced incorporation. We assume that a particular series of events or set of enzymatic conditions constitute a DNA synthesis-like (S-like) environment within the cell and that normal synthesis of DNA begins when the S-like environment is established and continues until replication is complete. The data presented in this paper and a previous paper [7] fit the following concept. 1 The average grain number (23.4) start of telophase until preparations an X-ray exposure of 512 r. Average tion = 16-18; 512 r = 13-16.
has been normalized to 35.4 min, the average time from the were fixed. The experiment described was repeated with grain numbers per 14.6 p2 at 30 to 40 min were: no irradia-
Experimental
Cell Research
37
44
R. A. McGrath,
W. M. Leach and J. G. Carlson
When replication of DNA is finished, the S-like environment does not immediately disappear but is gradually reduced as neuroblasts progress beyond the S period. The period of reduction corresponds to the time during which mitotic delay or reversion can be induced by irradiation (from very early prophase until the critical stage) and also to the stages of S-ray-induced incorporation of 3HTdR derivatives into chromosomal DNA. (This may also be the period when repair of radiation damage occurs.) Once the cells progress to the critical stage in very late prophase, the S-like condition is reduced to such a level that X-ray-induced incorporation of 3HTdR cannot occur. No mitotic delay or reversion occurs after this stage. Whatever damage has been done remains, and may or may not be corrected at some later cell time. It is obvious that Y-ray-induced incorporation of 3HTdR derivatives into chromosomal DNA is related to intracellular environmental conditions. Our data imply in addition a correlation to cell stage at the time of radiation exposure. SUMMARY
Autoradiographic studies show that grasshopper neuroblasts, trapped in mid-mitotic stages by colchicine and then exposed to 64 to 75,000 r of X-rays do not incorporate 3HTdR or its derivatives into DNA. The intracellular pool of TdR derivatives is not detectably changed by exposure to S-rays. The data show a correlation between radiation-induced incorporation of 3HTdR derivatives and cell stage at the time of irradiation. It is suggested that the synthesis-like condition, during which S-ray-induced incorporation of DN;A precursors can occur, degenerates gradually after cells stop normal synthesis of DNA. The comments and criticisms of Drs R. B. Setlow, R. F. Kimball and D. M. Prescott and discussions with them of various aspects of this work are gratefully acknowledged. Also we thank Dr S. Wolff for providing the colchicine used in this study. REFERENCES 1. CARLSON, J. G., Cold Spring Harbor Symp. 9, 104 (1941). 2. CARLSOX, J. G. and GAULDEN, M. E., in D. M. PRESCOTT (ed.) Methods in Cell Physiology, vol: 1. Academic Press, New York, 1964. 3. CARLSON, J. G. and HOLLAENDER, A., J. Cellular Comp. Physiol. 31, 149 (1948). 4. GAULDEN, M. E., Genetics 41, 645 (1956). 5. GAULDEN, M. E. and CARLSON, J. G., Expf! Cell Res. 2, 416 (1951). 6. LEACH. W. M.. Am. Sot. Biol. Bull. 10, 32 (1963). 7. MCGR~TH, R. A., Radiation Res. 19, 526 (1663). ’ 8. MCGRATH. R. A.. LEACH. W. M. and CARLSON, J. G., J. Roy. Microscop. Sot. 82, 55 (1963). 9. SHAW, E’I., Ex&Z Cell Res. 11, 580 (1956)
Experimental
Cell Research
37