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Sensitivity of DNA synthetic phase to near-ultraviolet radiation: Chromatid damage at early and late replication periods
Cancer Letters, 10 (1980) 253-259 o Elsevier/North-Holland Scientific Publishers Ltd.
253
SENSITIVITY OF DNA SYNTHETIC PHASE TO NEAR-ULTRAVIOLET RADIATION: CHROMATID DAMAGE AT EARLY AND LATE REPLICATION PERIODS
P. PANTAZIS Laboratory of Molecular Pharmacology, Health, Bethesda, MD 20205 (USA)
National Cancer Institute, National Institutes
of
(Received 16 May 1980) (Accepted 23 May 1980)
SUMMARY
Syrian hamster embryo (SHE) cells were pulse-labeled with bromodeoxyuridine at various periods of the S-phase, and then irradiated with near ultraviolet (NUV) light. Observation of the radiated chromosomes in the subsequent metaphase stage revealed that the period between the second and the fourth hour of the S-phase is most sensitive to radiation damage. The prevalent types of chromatid aberrations are ‘gaps’ and ‘breaks’. The results strongly suggest that the detrimental effect of NUV-radiation on chromosomes depends primarily on the particular stage when radiation is applied.
INTRODUCTION
It is well established that in many instances carcinogenesis is associated with the perturbation of DNA. For example, in the leukemia-prone genetic disorders of Bloom’s, Down’s, Fanconi’s syndromes and in ataxia-telangiectasia, there are increased frequencies of chromosome breaks and exchanges [l--5]. These aberrations are of the chromatid type, indicating that the damage was induced in the S- and/or GB-phases. Presumably, such aberrations are caused by various agents which exert a direct or indirect effect on the DNA of chromosomes during their replication. Several investigators have shown that replication of chromosomes is asynchronous but programmed during the S-phase of the cell cycle [ 6-101. Moreover, DNA synthesis, in any cell type, progresses from early- to latereplicating DNA, and whole chromosomes as well as regions of chromosomes can be either early- or late-replicating [ 11-131. In vitro, a many-fold increase of breaks can be produced in cellular DNA when incorporation of 5-bromodeoxyuridine (BUdR) in place of its analog
254
deoxythymidine (TdR) is followed by radiation with NUV-light, i.e. wavelengths around 300 nm [ 141. Syrian hamster embryo (SHE) cells are widely used for quantitative studies of neoplastic transformation induced by chemical carcinogens [ 15,161. In this study, synchronized SHE cells were pulse-labeled with BUdR at various periods of the S-phase, followed by radiation with NUVlight. The sensitivity of each period of the S-phase to NUV-radiation was measured as a function of the extent of chromosomal damage observed at the 1st metaphase (Ml) subsequent to the radiated S-phase. MATERIALS
AND METHODS
Ceil treatment SHE cells were established and cultured as described by Tsutsui et al. [17]. Briefly, logarithmically growing cells at a density of 2 X 10’ cells/ml were synchronized with 0.35 mM hydroxyurea (HU) for 12 h. After release from the HU-block (O-time), the Gl/S-arrested cells proceeded through the S-phase and were subjected to a l-h treatment with BUdR as follows: The S-phase was divided into five l-h periods. In each period, the cells were treated, in the dark, with medium containing BUdR in place of TdR. After 1 h of BUdR treatment, the cells, brought up in phosphate-buffered saline (PBS), were radiated with NUV-light [18]. PBS was replaced with TdRcontaining medium and the cells were allowed to proceed toward metaphase. Five hours after O-time, colcemid (0.2 pg/ml) was added to cultures for 4 h before the cells were harvested for microscopic observations. Preparation of chromosomal spreads Cells were harvested after trypsinization, fixed in Camoy’s fixative, and chromosomal spreads were prepared on flame-dried microscopic slides. Chromosomes were stained with Giemsa made in phosphate buffer, pH 6.8. RESULTS
Effects of various treatments on S-phase To study the effect of NUV-light at various periods of the S-phase using the methodology described above, it was necessary that BUdR treatment did not alter the cell cycle of, or cause cytogenetic effects on SHE cells. The effect of individual treatment on the cells in metaphase that accumulated during the 4-h period of colcemid treatment was determined as described in Table 1. About 25% of the non-synchronized-untreated (control) total cell population was arrested in metaphase under the experimental conditions used. The concentration of BUdR used in the present study had no cytotoxic affect on SHE cells since no changes in the percentages of metaphases were observed (Table 1). These results are consistent with previously reported findings that BUdR at this concentration (0.01 mM) did not disturb
255 TABLE 1 EFFECT OF BUdR AND NUV-LIGHT TREATMENT IN METAPHASE AT Ml
ON THE NUMBER OF CELLS
Treatment
Metaphase score (%)
[k\ (c)
BUdR concention used (mM)
Irradiation period (min )
0 0.01 0
0 0 30
26 (1607) 25 (3051) 10 (3422)
Non-synchronized SHE cells were seeded in 3 petri-dishes. In petri-dish (b) the cells were grown in BUdR-containing medium, whereas, in petri-dishes (a) and (c) the cells were grown in normal medium. Growth was allowed to proceed for 1 h before the cells were brought up in PBS. Cell in the petri-dish (c) were radiated with NUV-light. Cells in all dishes were brought up in normal medium containing colcemid. After 4 h, cells were harvested and cells in metaphase scored.
the normal cycling progression of synchronized cells [18]. Further, the detrimental action of NUV-light was assayed by radiating non-synchronizeduntreated SHE cells for various time periods. The percentage of cells in metaphase observed at Ml decreased as the duration of the radiation period increased (data not shown). Thus, NUV-radiation of non-synchronized cells for 30 min resulted in a dramatic decrease of the number of cells in metaphase, i.e. the metaphase score was only 40% of the metaphase score of the control cells (Table 1). A higher decrease of cells in metaphase was observed when BUdR treatment preceded the radiation by NUV-light. The effect of NUV-radiation period of BUdR treated cells is shown in Fig. 1. Radiation for 5 min does not effect the number of cells in metaphase. However, the longer the cells were radiated with NUV-light, the lower the number of observed cells in metaphase, with the lowest number of cells in metaphase (3%) observed after 30 min of radiation. Cy togene tic effects
Previous studies showed that incorporation of BUdR causes several highly specific breakpoints in Chinese hamster [19] and cactus mouse [ZO] chromosomes. Treatment of SHE cells with 0.01 mM BUdR for 1 h, at any period of the S-phase, did not result in a cytogenetic effect. However, BUdRtreated SHE cells, when radiated with NUV-light for 30 min, exhibited chromosomal abnormalities at Ml. These abnormalities were of the chromatid type, i.e. abnormalities which indicated that they occurred during the S-phase. In most cases, more than one aberration was observed per metaphase spread. A metaphase spread with representative chromatid aberrations is shown in Fig. 2. The results of the aberrations observed are summarized in Table 2. A high incidence of chromatid aberration was observed at the
256
Fig. 1. Correlation between irradiation period of the SHE cells and number of cells in metaphase observed at Ml. Non-synchronized cells were treated with 0.01 mM BUdR for 1 h prior to radiation with NUV-light, as described in ‘Materials and Methods’. The figures in parentheses indicate the number of cells screened after each irradiation period.
257
b(37051 O-_L-_L_L 10 IRRADIATION
20 PERIOD (mid
33
Fig. 2. Metaphase spreads of untreated (upper) and BUdR-NUV cells. Chromatid aberrations are circled.
treated (lower) SHE
2nd period of the S-phase. Gaps, breaks, and exchanges were the aberrations observed with a prevalence of gaps. No exchanges were observed at periods 4and5. DISCUSSION
Following BUdR treatment, the synchronized SHE cells were radiated with NUV-light at various periods of the S-phase, and chromatid aberrations were observed at Ml. The prevalence of chromatid gaps observed is a finding perhaps characteristic for such a combined treatment, since other treatments TABLE 2 CHROMATID ABERRATIONS OBSERVED IN SHE CELLS TREATED WITH BUdR AND RADIATED WITH NUV-LIGHT AT VARIOUS PERIODS OF THE S-PHASE Period of S-phase during which SHE cells were labeled with BUdR
Metaphases scored
l:O-lh 2:1-2h 3:2-3h 4: 3-4h 5:4-5h
132 226 100 113 111
Chromatid aberrations observed (%) Gaps
Breaks
Exchanges
6 48 20 20 8
8 24 6 10 3
8 15 6 0 0
SHE cells released from the HU-block (O-time) were treated with BUdR-containing medium for 1 h, for the periods indicated above. At the end of the BUdR-treatment the cells were radiated for 30 min as described in ‘Materials and Methods’, and the cells were harvested at Ml to score aberrations.
258
such as ionizing radiation or chemical carcinogens result mostly in chromosomal breaks [21]. Additionally, exchanges were observed only at the lst, 2nd, and 3rd periods of the S-phase. This suggests that the experimental conditions used in the present study favor the formation of exchanges only at the first 3 h of the S-phase. A high number of chromatid aberrations occurred predominantly between the 2nd and 5th period, presumably when most of the chromosomal DNA was involved in replication. The lowest number of aberrations occurred at the 5th period indicating either that most chromosomes had completed their replication, or that the late replicating chromosomes or segments of chromosomes were less sensitive to the treatments applied. This latter explanation is supported by studies which have shown that the heterochromatic regions of chromosomes, i.e. the chromosomal regions which are most resistant to various treatments, are latereplicating [ 221. The present studies show that the sensitivity of a cell in the S-phase to NUV-radiation depends greatly on the specific period of the S-phase at which the cell is radiated. In other words, the cell sensitivity to NUV-light and the concomitant damage of its chromosomes depends on the number of, and/or the vital segments of chromosomes synthesized at the time of radiation. Thus, this methodology may prove to be useful for the study of detailed replication of human chromosomes in vitro, and for studying in vivo action of carcinogenic agents (radiation or chemicals) on DNA. ACKNOWLEDGEMENTS
This work was performed at the School of Hygiene, Division of Biophysics, The Johns Hopkins University. Thanks to Dr. T. Tsutsui for suggestions and technical assistance. REFERENCES 1 German, J., Archibald, R. and Bloom, D. (1965) Chromosomal breakage in a rare and probably genetically determined syndrome in man. Science, 148, 506-507. 2 Hecht, F., Koier, R., Rigq D., Dahnke, G., Case, M., Tisdale, V. and Miller, R. (1966) Leukemia and lymphocytes in ataxia-telangiectasia. Lancet, 2, 1193. 3 Sasaki, M.S. and Tonomura, A. (1973) A high susceptibility of Fanconi’s anemia to chromosome breakage by DNA cross-linking agents. Cancer Res., 33, 1829-1836. 4 Higurashi, M. and Cohen, P.E. (1973) In vitro chromosomal radiosensitivity in chromosomal breakage syndromes. Cancer, 32, 380-383. 5 Chaganti, R.S.K., Schomberg, S. and German, J. (1974) A many fold increase in sister chromatid exchanges in Bloom’s syndrome lymphocytes. Proc. Natl. Acad. Sci. USA, 71,4508-4512. 6 Taylor, J.H. (1960) Asynchronous duplication of chromosomes in cultured cells of Chinese hamster. J. Biophys. Biochem. Cytol., 7, 455-467. 7 Bader, S., Miller, O.J. and Mukherjee, B.B. (1963) Observations on chromosome duplication in cultured human leukocytes. Exp. Cell Res., 31, 100-112. 8 Kikuchi, Y. and Sandberg, A.A. (1964) Chronology and pattern of human chromosome replication. 1. Blood leukocytes of normal subjects. J. Natl. Cancer Inst., 32.1109-1143.
259
9 Braun, R. and Willi, H. (1966) Time sequence of DNA replication in Physarum. Biochim. Biophys. Acta, 174, 246-252. 10 Taylor, J.H., Myers, T.L. and Cunningham, H.L. (1971) Programmed synthesis of deoxyribonucleic acid during the cell cycle. In Vitro, 6, 309-321. 11 Gilbert, C.W., Muldal, S. and Lajtha, L.G. (1965) Rate of chromosome duplication at the end of the deoxyribonucleic acid synthetic period in human blood cells. Nature, 208,159-161. 12 Mueller, G.C. and Kajiwava, K. (1966) Early-and-late replicating deoxyribonucleic acid complexes in HeLa nuclei. Biochim. Biophys. Acta, 114, 180-115. 13 Gavosto, F., Pegogaro, L. Masera, P. and Rovera, G. (1968) Late DNA replication pattern in human haemopoietic cells. A comparative investigation using a high resolution quantitative autoradiography. Exp. Cell Res., 49,340-358. 14 Hutchinson, F. (1973) The lesions produced by ultraviolet light in DNA containing 5-bromouracil. Q. Rev. Biophys., 6, 201-246. 15 Berwald, Y. and Sachs, L. (1965). In uitro transformation of normal cells to tumor cells by carcinogenic hydrocarbons. J. Natl. Cancer Inst., 35, 641-661. 16 DiPaolo, J.A., Donovan, P. and Nelson, R. (1969) Quantitative studies of in vitro transformation by chemical carcinogens. J. Natl. Cancer Inst., 42, 867-874. 17 Barrett, J.C., Tsutsui, T. and Ts’o, P.O.P. (1978) Neoplastic transformation induced by a direct perturbation of DNA. Nature, 274, 229-232. 18 Tsutsui, T., Barrett, J.C. and Ts’o, P.O.P. (1979) Morphological transformation, DNA damage, and chromosomal aberrations induced by a direct perturbation of synchronized Syrian hamster embryo cells. Cancer Res., 39, 2356-2365. 19 Hsu, T.C. and Somers, C.E. (1961) Effect of 5-bromodeoxyuridine on mammalian chromosomes. Proc. Natl. Acad. Sci. USA, 47,396-403. 20 Schneider, N.R., Chaganti, R.S.K. and German, J. (1980) Analysis of a BrdUsensitive site in the cactus mouse (Peromyscus eremicus): Chromosomal breakage and sister-chromatid exchange. Chromosoma, 77, 379-389. 21 Hsu, T.C. and Au, W. (1979) Effects of chemical carcinogens on chromosomes. In: Effects of Drugs on the Cell Nucleus, Vol. 1, pp. 315-332. Editors: H. Busch, S.T. Crooke and Y. Daskal. Bristol-Myers Cancer Symposia. Academic Press, New York. 22 Lima-de-Faria, A, (1969) DNA replication and gene amplification in heterochromatin. In: Handbook of Molecular Cytology, pp. 278-325. Editor: A. Lima-de-Faria. North-Holland Publishing Company, Amsterdam.
253
SENSITIVITY OF DNA SYNTHETIC PHASE TO NEAR-ULTRAVIOLET RADIATION: CHROMATID DAMAGE AT EARLY AND LATE REPLICATION PERIODS
P. PANTAZIS Laboratory of Molecular Pharmacology, Health, Bethesda, MD 20205 (USA)
National Cancer Institute, National Institutes
of
(Received 16 May 1980) (Accepted 23 May 1980)
SUMMARY
Syrian hamster embryo (SHE) cells were pulse-labeled with bromodeoxyuridine at various periods of the S-phase, and then irradiated with near ultraviolet (NUV) light. Observation of the radiated chromosomes in the subsequent metaphase stage revealed that the period between the second and the fourth hour of the S-phase is most sensitive to radiation damage. The prevalent types of chromatid aberrations are ‘gaps’ and ‘breaks’. The results strongly suggest that the detrimental effect of NUV-radiation on chromosomes depends primarily on the particular stage when radiation is applied.
INTRODUCTION
It is well established that in many instances carcinogenesis is associated with the perturbation of DNA. For example, in the leukemia-prone genetic disorders of Bloom’s, Down’s, Fanconi’s syndromes and in ataxia-telangiectasia, there are increased frequencies of chromosome breaks and exchanges [l--5]. These aberrations are of the chromatid type, indicating that the damage was induced in the S- and/or GB-phases. Presumably, such aberrations are caused by various agents which exert a direct or indirect effect on the DNA of chromosomes during their replication. Several investigators have shown that replication of chromosomes is asynchronous but programmed during the S-phase of the cell cycle [ 6-101. Moreover, DNA synthesis, in any cell type, progresses from early- to latereplicating DNA, and whole chromosomes as well as regions of chromosomes can be either early- or late-replicating [ 11-131. In vitro, a many-fold increase of breaks can be produced in cellular DNA when incorporation of 5-bromodeoxyuridine (BUdR) in place of its analog
254
deoxythymidine (TdR) is followed by radiation with NUV-light, i.e. wavelengths around 300 nm [ 141. Syrian hamster embryo (SHE) cells are widely used for quantitative studies of neoplastic transformation induced by chemical carcinogens [ 15,161. In this study, synchronized SHE cells were pulse-labeled with BUdR at various periods of the S-phase, followed by radiation with NUVlight. The sensitivity of each period of the S-phase to NUV-radiation was measured as a function of the extent of chromosomal damage observed at the 1st metaphase (Ml) subsequent to the radiated S-phase. MATERIALS
AND METHODS
Ceil treatment SHE cells were established and cultured as described by Tsutsui et al. [17]. Briefly, logarithmically growing cells at a density of 2 X 10’ cells/ml were synchronized with 0.35 mM hydroxyurea (HU) for 12 h. After release from the HU-block (O-time), the Gl/S-arrested cells proceeded through the S-phase and were subjected to a l-h treatment with BUdR as follows: The S-phase was divided into five l-h periods. In each period, the cells were treated, in the dark, with medium containing BUdR in place of TdR. After 1 h of BUdR treatment, the cells, brought up in phosphate-buffered saline (PBS), were radiated with NUV-light [18]. PBS was replaced with TdRcontaining medium and the cells were allowed to proceed toward metaphase. Five hours after O-time, colcemid (0.2 pg/ml) was added to cultures for 4 h before the cells were harvested for microscopic observations. Preparation of chromosomal spreads Cells were harvested after trypsinization, fixed in Camoy’s fixative, and chromosomal spreads were prepared on flame-dried microscopic slides. Chromosomes were stained with Giemsa made in phosphate buffer, pH 6.8. RESULTS
Effects of various treatments on S-phase To study the effect of NUV-light at various periods of the S-phase using the methodology described above, it was necessary that BUdR treatment did not alter the cell cycle of, or cause cytogenetic effects on SHE cells. The effect of individual treatment on the cells in metaphase that accumulated during the 4-h period of colcemid treatment was determined as described in Table 1. About 25% of the non-synchronized-untreated (control) total cell population was arrested in metaphase under the experimental conditions used. The concentration of BUdR used in the present study had no cytotoxic affect on SHE cells since no changes in the percentages of metaphases were observed (Table 1). These results are consistent with previously reported findings that BUdR at this concentration (0.01 mM) did not disturb
255 TABLE 1 EFFECT OF BUdR AND NUV-LIGHT TREATMENT IN METAPHASE AT Ml
ON THE NUMBER OF CELLS
Treatment
Metaphase score (%)
[k\ (c)
BUdR concention used (mM)
Irradiation period (min )
0 0.01 0
0 0 30
26 (1607) 25 (3051) 10 (3422)
Non-synchronized SHE cells were seeded in 3 petri-dishes. In petri-dish (b) the cells were grown in BUdR-containing medium, whereas, in petri-dishes (a) and (c) the cells were grown in normal medium. Growth was allowed to proceed for 1 h before the cells were brought up in PBS. Cell in the petri-dish (c) were radiated with NUV-light. Cells in all dishes were brought up in normal medium containing colcemid. After 4 h, cells were harvested and cells in metaphase scored.
the normal cycling progression of synchronized cells [18]. Further, the detrimental action of NUV-light was assayed by radiating non-synchronizeduntreated SHE cells for various time periods. The percentage of cells in metaphase observed at Ml decreased as the duration of the radiation period increased (data not shown). Thus, NUV-radiation of non-synchronized cells for 30 min resulted in a dramatic decrease of the number of cells in metaphase, i.e. the metaphase score was only 40% of the metaphase score of the control cells (Table 1). A higher decrease of cells in metaphase was observed when BUdR treatment preceded the radiation by NUV-light. The effect of NUV-radiation period of BUdR treated cells is shown in Fig. 1. Radiation for 5 min does not effect the number of cells in metaphase. However, the longer the cells were radiated with NUV-light, the lower the number of observed cells in metaphase, with the lowest number of cells in metaphase (3%) observed after 30 min of radiation. Cy togene tic effects
Previous studies showed that incorporation of BUdR causes several highly specific breakpoints in Chinese hamster [19] and cactus mouse [ZO] chromosomes. Treatment of SHE cells with 0.01 mM BUdR for 1 h, at any period of the S-phase, did not result in a cytogenetic effect. However, BUdRtreated SHE cells, when radiated with NUV-light for 30 min, exhibited chromosomal abnormalities at Ml. These abnormalities were of the chromatid type, i.e. abnormalities which indicated that they occurred during the S-phase. In most cases, more than one aberration was observed per metaphase spread. A metaphase spread with representative chromatid aberrations is shown in Fig. 2. The results of the aberrations observed are summarized in Table 2. A high incidence of chromatid aberration was observed at the
256
Fig. 1. Correlation between irradiation period of the SHE cells and number of cells in metaphase observed at Ml. Non-synchronized cells were treated with 0.01 mM BUdR for 1 h prior to radiation with NUV-light, as described in ‘Materials and Methods’. The figures in parentheses indicate the number of cells screened after each irradiation period.
257
b(37051 O-_L-_L_L 10 IRRADIATION
20 PERIOD (mid
33
Fig. 2. Metaphase spreads of untreated (upper) and BUdR-NUV cells. Chromatid aberrations are circled.
treated (lower) SHE
2nd period of the S-phase. Gaps, breaks, and exchanges were the aberrations observed with a prevalence of gaps. No exchanges were observed at periods 4and5. DISCUSSION
Following BUdR treatment, the synchronized SHE cells were radiated with NUV-light at various periods of the S-phase, and chromatid aberrations were observed at Ml. The prevalence of chromatid gaps observed is a finding perhaps characteristic for such a combined treatment, since other treatments TABLE 2 CHROMATID ABERRATIONS OBSERVED IN SHE CELLS TREATED WITH BUdR AND RADIATED WITH NUV-LIGHT AT VARIOUS PERIODS OF THE S-PHASE Period of S-phase during which SHE cells were labeled with BUdR
Metaphases scored
l:O-lh 2:1-2h 3:2-3h 4: 3-4h 5:4-5h
132 226 100 113 111
Chromatid aberrations observed (%) Gaps
Breaks
Exchanges
6 48 20 20 8
8 24 6 10 3
8 15 6 0 0
SHE cells released from the HU-block (O-time) were treated with BUdR-containing medium for 1 h, for the periods indicated above. At the end of the BUdR-treatment the cells were radiated for 30 min as described in ‘Materials and Methods’, and the cells were harvested at Ml to score aberrations.
258
such as ionizing radiation or chemical carcinogens result mostly in chromosomal breaks [21]. Additionally, exchanges were observed only at the lst, 2nd, and 3rd periods of the S-phase. This suggests that the experimental conditions used in the present study favor the formation of exchanges only at the first 3 h of the S-phase. A high number of chromatid aberrations occurred predominantly between the 2nd and 5th period, presumably when most of the chromosomal DNA was involved in replication. The lowest number of aberrations occurred at the 5th period indicating either that most chromosomes had completed their replication, or that the late replicating chromosomes or segments of chromosomes were less sensitive to the treatments applied. This latter explanation is supported by studies which have shown that the heterochromatic regions of chromosomes, i.e. the chromosomal regions which are most resistant to various treatments, are latereplicating [ 221. The present studies show that the sensitivity of a cell in the S-phase to NUV-radiation depends greatly on the specific period of the S-phase at which the cell is radiated. In other words, the cell sensitivity to NUV-light and the concomitant damage of its chromosomes depends on the number of, and/or the vital segments of chromosomes synthesized at the time of radiation. Thus, this methodology may prove to be useful for the study of detailed replication of human chromosomes in vitro, and for studying in vivo action of carcinogenic agents (radiation or chemicals) on DNA. ACKNOWLEDGEMENTS
This work was performed at the School of Hygiene, Division of Biophysics, The Johns Hopkins University. Thanks to Dr. T. Tsutsui for suggestions and technical assistance. REFERENCES 1 German, J., Archibald, R. and Bloom, D. (1965) Chromosomal breakage in a rare and probably genetically determined syndrome in man. Science, 148, 506-507. 2 Hecht, F., Koier, R., Rigq D., Dahnke, G., Case, M., Tisdale, V. and Miller, R. (1966) Leukemia and lymphocytes in ataxia-telangiectasia. Lancet, 2, 1193. 3 Sasaki, M.S. and Tonomura, A. (1973) A high susceptibility of Fanconi’s anemia to chromosome breakage by DNA cross-linking agents. Cancer Res., 33, 1829-1836. 4 Higurashi, M. and Cohen, P.E. (1973) In vitro chromosomal radiosensitivity in chromosomal breakage syndromes. Cancer, 32, 380-383. 5 Chaganti, R.S.K., Schomberg, S. and German, J. (1974) A many fold increase in sister chromatid exchanges in Bloom’s syndrome lymphocytes. Proc. Natl. Acad. Sci. USA, 71,4508-4512. 6 Taylor, J.H. (1960) Asynchronous duplication of chromosomes in cultured cells of Chinese hamster. J. Biophys. Biochem. Cytol., 7, 455-467. 7 Bader, S., Miller, O.J. and Mukherjee, B.B. (1963) Observations on chromosome duplication in cultured human leukocytes. Exp. Cell Res., 31, 100-112. 8 Kikuchi, Y. and Sandberg, A.A. (1964) Chronology and pattern of human chromosome replication. 1. Blood leukocytes of normal subjects. J. Natl. Cancer Inst., 32.1109-1143.
259
9 Braun, R. and Willi, H. (1966) Time sequence of DNA replication in Physarum. Biochim. Biophys. Acta, 174, 246-252. 10 Taylor, J.H., Myers, T.L. and Cunningham, H.L. (1971) Programmed synthesis of deoxyribonucleic acid during the cell cycle. In Vitro, 6, 309-321. 11 Gilbert, C.W., Muldal, S. and Lajtha, L.G. (1965) Rate of chromosome duplication at the end of the deoxyribonucleic acid synthetic period in human blood cells. Nature, 208,159-161. 12 Mueller, G.C. and Kajiwava, K. (1966) Early-and-late replicating deoxyribonucleic acid complexes in HeLa nuclei. Biochim. Biophys. Acta, 114, 180-115. 13 Gavosto, F., Pegogaro, L. Masera, P. and Rovera, G. (1968) Late DNA replication pattern in human haemopoietic cells. A comparative investigation using a high resolution quantitative autoradiography. Exp. Cell Res., 49,340-358. 14 Hutchinson, F. (1973) The lesions produced by ultraviolet light in DNA containing 5-bromouracil. Q. Rev. Biophys., 6, 201-246. 15 Berwald, Y. and Sachs, L. (1965). In uitro transformation of normal cells to tumor cells by carcinogenic hydrocarbons. J. Natl. Cancer Inst., 35, 641-661. 16 DiPaolo, J.A., Donovan, P. and Nelson, R. (1969) Quantitative studies of in vitro transformation by chemical carcinogens. J. Natl. Cancer Inst., 42, 867-874. 17 Barrett, J.C., Tsutsui, T. and Ts’o, P.O.P. (1978) Neoplastic transformation induced by a direct perturbation of DNA. Nature, 274, 229-232. 18 Tsutsui, T., Barrett, J.C. and Ts’o, P.O.P. (1979) Morphological transformation, DNA damage, and chromosomal aberrations induced by a direct perturbation of synchronized Syrian hamster embryo cells. Cancer Res., 39, 2356-2365. 19 Hsu, T.C. and Somers, C.E. (1961) Effect of 5-bromodeoxyuridine on mammalian chromosomes. Proc. Natl. Acad. Sci. USA, 47,396-403. 20 Schneider, N.R., Chaganti, R.S.K. and German, J. (1980) Analysis of a BrdUsensitive site in the cactus mouse (Peromyscus eremicus): Chromosomal breakage and sister-chromatid exchange. Chromosoma, 77, 379-389. 21 Hsu, T.C. and Au, W. (1979) Effects of chemical carcinogens on chromosomes. In: Effects of Drugs on the Cell Nucleus, Vol. 1, pp. 315-332. Editors: H. Busch, S.T. Crooke and Y. Daskal. Bristol-Myers Cancer Symposia. Academic Press, New York. 22 Lima-de-Faria, A, (1969) DNA replication and gene amplification in heterochromatin. In: Handbook of Molecular Cytology, pp. 278-325. Editor: A. Lima-de-Faria. North-Holland Publishing Company, Amsterdam.