J. Photo&em.
Photobiol.
B: Biol., 9 (1991)
171-179
171
Changes in cell cycle distribution of V79 Chinese hamster fibroblasts after irradiation at different wavelengths F. Z61zert, B. Heil and J. Kiefer Strahlenzentrum ok Justus-Liebig-Universitfit 6300 G&&n (F.R. G.]
Gi@en,
Leihgesterner
Weg 217,
(Received August 28, 1990; accepted September 10, 1990)
Keywords. UV effects, flow cytometry, DNA damage, pyrimidine dimers.
Abstract Changes in celI cycle distribution of V79 Chinese hamster fibroblasts were investigated at different wavelengths between 254 and 313 run. The fluences applied led to surviving fractions of 0.61. In all cases, the S fraction was temporarily increased within 8-12 h after irradiation, whereas the G1 fraction was decreased. The maximum deviations from the initial values did not signifkantly depend on the wavelength.
1. Introduction During the last few years the wavelength dependences of many different UV radiation effects have been investigated. In bacteria, for instance, action spectra are known for membrane damage [ 1, 21 and the destruction of different transport systems [3-61 as well as for growth delay [ 7, 81. Research with eukaryotes at the same time has been largely restricted to inactivation and mutation induction. There are reports on membrane damage and growth delay in yeast [9] and the inhibition of DNA synthesis in frog cells [lo], but no comparable data for mammalian cells have been published. Thus the present study of cell cycle disturbances in Chinese hamster fibroblasts may be of particular interest. 2. Materials and methods 2.1. Culture cond&kms Chinese hamster fibroblasts V79A received from M. Fox, Manchester, were used for this study. Cultures were grown at 37 “C in Dulbecco’s Modified +Author to whom correspondence should be addressed at: Institut fiir Medizinische Strahlenbiologie, Universit&tskhnikumEssen, Hufelandstrai3e 55, 4300 Essen, F.R.G.
loll-1344/91/$3.50
Q Elsevier Sequoia/Printed in The Netherlands
172
Eagle Medium with 4500 mg I-’ glucose without sodium pyruvate (Gibco, Paisley, U.K.) supplemented with 5% foetal calf serum (Gibco). IV-[2-Hydroxyethyllpiperazine-IV’-[ 2-ethane-sulphonic acid] (HEPES) buffer (1 M; 1 ml 1-l; Seromed, Berlin, F.R.G.), Penicillin (0.06 g 1-l) and streptomycin (0.05 g 1-l) were added to the medium. Cells were subcultured every 3-4 days and regularly checked for mycoplasm. For the experiments, cells from a confhrent culture were plated at a density of lo5 ml-’ on several culture dishes (diameter, 9 cm) containing 10 ml medium and incubated overnight. Before irradiation the medium was withdrawn, traces were washed away with about 10 ml phosphate-buffered saline (PBS) (8 g NaCI, 2 g KCl, 1.5 g Na&IP04, 0.2 g KHpPOl per litre) and 10 ml PBS was added per dish. The cells were then irradiated as described below, after which the buffer was again replaced by 10 ml medium. After incubation for different times, the cells were trypsinized, centrifuged (lOOg, 5 ruin) and fixed in 70% ethanol. 2.2. F7ow fq_mmf?t?y For the staining procedure the cells were again centrifuged (2OOg, 5 mm) and resuspended in 2 ml pepsin solution (0.5 g pepsin (1000 U g-l, Serva), 5.5 ml 1 N HCI and 100 ml distilled water, pH 1.8). After 5 min, tris buffer (12 g tris-(hydroxymethyl)aminomethane, 6 g NaCl, 84 ml 1 N HCl and 1000 ml distilled water, pH 7.5) was added to stop the enzyme reaction. The cells were centrifuged for a third time (2OOg, 5 min), left with 6-8 drops of ribonuclease solution (0.1 g ribonuclease (50 U mg- ‘, Boehringer) in 100 ml distilled water) for 5 min and then resuspended in 4 ml ethidiumbromide-mithramycin solution (1 mg ethidiumbromide, 2.5 mg mithramycin, 305 mg MgClz.6H20 in 100 ml tris buffer). Measurements were performed with a flow cytometer (ICP 22, Phywe). 2.3. Radiation sources A 10 W low pressure mercury lamp (HNS 12, Osram) for 254 run and a 1000 W mercury arc lamp (1000 mercury (xenon), Oriel) equipped with a prism monochromator (designed by Meyer, 1966) for wavelengths between 265 and 313 m-nwere used. The setting of the monochromator was checked with the aid of a spectrophotometer (PMQ 3, Zeiss). The bandwidth (full width at half-maximum) of the radiation was between 2.5 and 4.0 run (see Table 1). As the culture dishes had to be irradiated in a horizontal position, the beam from the monochromator was deflected by a mirror. The resulting radiation field was somewhat inhomogeneous; therefore, the culture dishes were acentrically rotated to ensure as uniform an energy distribution as possible. Instead of the normal plastic lid a quartz cover was used for the culture dishes. The fluence rates were determined separately for each experiment by potassium ferrioxalate actinometry [ 111 (see Table 1). 3. Results The measurements yielded histograms showing the number of cells at different DNA contents. There were normally two clearly distinguishable
173 TABLE
1
Parameters
of irradiation
Wavelength
Bandwidth
Fluence
(nm)
(nm)
(w mm2)
Fluence (J ms2)
254 265 273 283 293 303 313
2.7 3.0 3.3 3.7 3.9 3.1
0.41 0.27 0.12 0.13 0.52 0.45 1.66
5.7 4.5 4.5 5.2 17.1 323.0 6940.0
rate
applied
maxima which could be attributed to the G1 and Gz fractions. For a quantitative evaluation of the histograms, certain assumptions had to be made about the S fraction. We chose two methods of analysis: graphical and computational. In the first case, the S fraction was represented by a rectangle extending from the middle of the G1 peak to the middle of the G2 peak; the areas left and right of the rectangle were taken for the Gr and Ga fractions respectively [ 121. In the second case, the S fraction was approximated by a broadened polynomial and the G1 and Gz fractions by gaussian curves; the sum of these three functions was fitted to the actual distribution by a least-squares procedure 1131. Several studies have shown that the latter method leads to rather accurate results for exponentially growing cultures, as judged by comparison with autoradiographical data [ 141. However, a UV irradiated culture is certainly not growing exponentially and the computational fit was rather unsatisfactory for some of our histograms. We could have added another gaussian curve with variable position to the S fraction [ 151, but that would have greatly increased the computation time. Moreover, it seems that the conclusions drawn do not depend on the specific method of histogram evaluation. The data from the computational analysis are given here; the graphical method leads to very similar results. Changes in the cell cycle distribution were investigated after irradiation at seven different wavelengths which had been used in earlier studies of inactivation (loss of colony forming ability) [ 161. The fluences applied led to surviving fractions of 0.61, i.e. the mean number of “lethal hits” per cell was 0.5. In some cases, the values in Table 1 do not agree with those previously published, since later experiments required certain corrections. However, substantial differences only occurred at 303 nm (323 instead of 165 J rne2) and 313 run (6940 instead of 4170 J mb2). This was due to a reduction in stray light as a consequence of recoating the monochromator mirrors. Samples were taken at 4 h intervals up to 24 h after irradiation. Figure 1 shows a typical series of cell cycle distributions obtained in this way. Each experiment was carried out two to four times, and for each cell cycle
0
100
50
ct!ArwL
150
MJMBER
BOO u b
600
400 200
0 0
Yl
100
150
CHAFNELW-lBER
,000
I
1
Fig. 1. Changes in cell cycle distribution after irradiation at 254 nm. Typical series of histograms obtained at different time intervals. Calculated distributions for G1, S and Gz phases together with the sum of all three functions (-).
175
distribution two histograms were recorded. The G1, S and G2 fractions and their difterences from the initial values were determined. Figure 2 and Table 2 summarize the data obtained. All quantities given are means and the standard errors were obtained from interexperimental variation. Regardless of the wavelength applied, similar changes in cell cycle distribution were observed. After irradiation, the fraction of cells was temporarily increased in the early S phase (4-8 h after irradiation), in the middle S phase (8-12 h after irradiation) and in the late S phase (12-16 h after irradiation). On the whole, the S fraction reached a maximum after 8-12 h, while the G, fraction reached a minimum at the same time. These changes appeared somewhat earlier after short wavelength than after long wavelength irradiation. However, the maximal deviations from the initial values were essentially the same in all cases. When the cells were not irradiated, but otherwise treated in the same manner as described above, no significant changes in cell cycle distribution occurred. The experimental conditions in the absence of irradiation obviously had no effect on cell proliferation. 4. Discussion Changes in cell cycle distribution after UV irradiation at 254 run have been investigated by Imray et al. [ 171 in human lymphoblastoid cells and by KiriIlova et al. [ 18, 191 in V79 Chinese hamster fibroblasts. Both studies support earlier evidence from work with synchronized cultures [20-231 that, in normal cells, only the S phase is delayed, whereas the G1 and G2 phases are not affected. However, in certain repair deficient strains, the passage from G1 to S is also inhibited. The latter effect seems to be related to a low ability of the cells to seal gaps which occur during excision repair or replication of a damaged template (human cells: XP variant; Chinese hamster cells: CHS 2). Our results are in good agreement with those of Imray et al. and Kirillova et al. and further corroborate their conclusions, albeit on different grounds. We were primarily interested in the wavelength dependence of cell cycle disturbances. It has been suggested earlier that, between 254 and 313 nm, the inactivation of Chinese hamster cells correlates with the formation of pyrimidine dimers [ 241. In a more detailed analysis, however, such a correlation was only found for the final slope of the survival curves, whereas the shoulder of the survival curves seemed to be more pronounced at longer wavelengths than at shorter wavelengths [ 16, 251. For the experiments described here, we used fluences which reduced the surviving fraction to 0.61, i.e. almost identical to the shoulder width of the survival curves. Had we sought to induce the same number of dimers in each case, we would have applied 50% smaller fluences at 303 and 313 run. Indeed, a number of earlier experiments were carried out in this way. Since we did not have a suitable computer program at our disposal, the
176
I-
I
0.
4
12
.3
TIME
0
4
8 TIME
16
20
IL.-I
Ih
12 , h
16
20
2L
-7--
__,
A 0
1.
8
0
&
8
12 TIME I h
4
8
IIME
2. Changes in cell cycle distribution after irradiation at 254313 G1 (El), S (0) and Gz (A) fractions. Fig.
20
24
16
20
21
.--A TIME I
0
J 16
A2
12 I h
16
20
24
ruu. Mean values for
177 TABLE
2
Overall effect of irradiation Wavelength 0-w
254 265 273 283 293 303 313 “Sum of deviations
on the cell cycle
Maximal deviation from initial value
Sum of deviations from initial value”
G, (96)
s (%I
G
-21.7k5.5 - 19.3 + 2.6 -20.1*6.6 -15.5k5.6 -21.5k3.5 -33.2*2.9 - 18.8 + 3.3
25.0 f 7.0 29.5 +2.0 21.Ok7.0 16.1 f5.1 19.0 f 6.8 21.6k2.3 17.3 f 8.3
-37.4* 14.2 - 34.8 f 8.2 -70.2k31.7 -41.6& 13.9 - 40.9 & 9.8 -49.6& 13.6 -41.0* 10.0
at 4, 8, 12,
(%I
s (46) 38.3 f 12.8 42.4+8.6 62.7536.7 41.0& 15.6 30.0 f 19.6 40.8 f 10.8 32.1 f 17.5
16 and 20 h.
data were analysed by graphical means only. It is clear, however, that the shift in the G1 and S fractions appeared at an earlier time after irradiation and the maximum deviation was reduced by a factor of about two compared with the results shown in Fig. 2 and Table 2. Thus it is not self-evident that we should observe similar changes in cell cycle distribution when fluences leading to the same survival level are chosen. Loss of colony forming ability is a comparatively late test parameter determined 7 days after irradiation, whereas the effects described here occur within the first 24 h. Nevertheless, there seems to be a correlation between the two end points, suggesting that changes in cell cycle distribution do not just depend on the number of pyrimidine dimers induced, but also on the ability of the cells to recover from sublethal damage. As mentioned above, Imray et al. and Kirillova et al. interpreted their results in much the same way. DNA lesions other than pyrimidine dimers are thought to play a role in the region above 300 run. The relative frequencies of glycols, strand breaks and DNA-protein crosslinks increase with wavelength [26-281. If these types of damage contribute to the effects on the cell cycle, we should not observe similar distributions with comparatively large numbers of dimers at 303 and 313 run, as was the case here, but just the opposite. Rosenstein [lo] reported a coincidence of the action spectra for inhibition of DNA synthesis and formation of endonuclease sensitive sites in frog cells. However, the photoreactive sectors seemed to be slightly reduced at wavelengths above 300 nm, pointing to a role for non-dimer damage. Since the cells used by Rosenstein were deficient in excision repair, the situation may be quite different from ours or, alternatively, inhibition of DNA synthesis may not be the only reason for cell cycle disturbances.
178
Acknowledgments
The cytofluorometrical measurements were performed in the laboratory of D. Kraushaar, Urological Clinic of the University of GiefSen, whose help is gratefully acknowledged. We thank B. Barth for excellent and reliable technical assistance and Dr. T. V. Kirillova for valuable comments on the manuscript. The investigations were supported by the “Bundesministerium fiir Forschung und Technologie” of the F.R.G. through the “Gesellschaft fiir Strahlen- und Umweltforschung” (KBF 49). References 1 L. R. Kelland, S. H. Moss and D. J. G. Davies, An action spectrum for ultraviolet radiation induced membrane damage in Escherichia coli K-12, Photo&em. Photobiol., 37 (1983)
307-312. 2 L. R. Kelland, S. H. Moss and D. J. G. Davies, Leakage of asRbf after ultraviolet irradiation of Eschmichia coli K-12, Photochem. Photobiol., 39 (1984) 329-336. 3 F. T. Robb, J. H. Hauman and M. J. Peak, Similar spectra for the inactivation by monochromatic light of two distinct leucine transport systems of Escherichia coli, Photo&em. Photobiol., 27 (1978) 465-469. 4 J. M. Ascenzi and J. Jagger, Ultraviolet action spectrum (238-405 nm) for inhibition of glycine uptake in E. coli, Photo&em, Photobiol., 30 (1979) 661-666. 5 F. T. Robb and M. J. Peak, Inactivation of the lactose permease of Escherichiu coli by monochromatic ultraviolet light, Photo&em, Photobtil., 30 (1979) 379-383. 6 R. C. Sharma and J. Jagger, Ultraviolet (254-405 run) action spectrum and kinetic studies of alanine uptake in Escherichia coli B/r, Photo&em. Photobiol., 33 (1981) 173-177. 7 J. Jagger, W. C. Wise and R. S. Stafford, Delay in growth and division induced by near ultraviolet radiation in Eschmichia coli B and its role in photoprotection and liquid holding recovery, Photochem. Photobiol., 3 (1964) 11-24. 8 H. E. Kubitschek and M. J. Peak, Action spectrum for growth delay induced by near ultraviolet light ln E. Coli B/r K, Photo&em. Photobiol., 31 (1980) 55-58. 9 J. Kiefer, M. Schall and A. Al-Talibi, Physiological responses of yeast cells to DV of different wavelengths, in R. C. Worrest (ed.), Stratospheric Ozone Reduction, Solar Ultraviolet Radiation and Plant Life, Springer, Berlin, 1986, pp. 151-159. 10 B. S. Rosenstem, Inhibition of semiconservative DNA synthesis in ICR 2A frog cells exposed to monochromatic UV wavelengths (252-313 nm) and photoreactivating light, Radiat. Res., 90 (1982) 509-517. 11 C. G. Hatchard and C. A. Parker, A new sensitive chemical actionometer. II. Potassium ferrioxalate as a standard chemical actionometer, Proc. R. Sot. London, Ser. A, 235 (1956) 518-536. 12 H. Baisch, W. Giihde and W. A. Linden, Analysis of PCP-data to determine the fraction of cells in the various phases of the cell cycle, Radiat. Environ. Biophys., I2 (1975) 3139. 13 P. N. Dean and J. H. Jett, Mathematical analysis of DNA distributions derived from flow microfluorometry, J. Cell. Biol., 60 (1974) 523-527. 14 J. W. Gray, P. N. Dean and M. L. Mendelsohn, Quantitative cell cycle analysis, in M. R. Melamed, P. F. Mullaney and M. L. Mendelsohn (eds.), Flow Cytometry and Sorting, Wiley, New York, 1979, pp. 383-407. 15 M. Fox, A model for the computer analysis of synchronous DNA distributions obtained by flow cytometry, Cytometry, I (1980) 71-77. 16 F. Ziilzer and J. Kiefer, Wavelength dependence of inactivation and mutation induction to 6-thioguanine resistance in V79 Chinese hamster ilbroblasts, Photochem. Photobiol., 40 (1984) 49-53.
179 17 P. Imray, T. Mangan, A. Saul and C. Kidson, Effects of ultraviolet irradiation on the ceII cycle in normal and W-sensitive ceII lines with reference to the nature of the defect in Xeroti pigmentosum variant, Mutat. Res., 112 (1983) 301-309. 18 T. V. Kiriiova, Y. M. Rozanov and I. M. Martynova, Effects of W-irradiation on the mitotic cycle in Chinese hamster cells with dierent W-sensitivity. I. Time course dependence following W-irradiation of cells, Tsitologiya, 27 (1985) 1285-1291 (in Russian). 19 T. V. Kiriilova, Y. M. Rozanov and I. M. Martynova, Effects of W-irradiation on the mitotic cycle in Chinese hamster cells with different W-sensitivity. II. Stathmokinetic studies of W-irradiated cultures, Tsitologiya, 27 (1985) 1380-1387 (in Russian). 20 B. Djordjevic and L. J. ToImach, Responses of synchronous populations of HeLa cells to ultraviolet radiation at selected stages of the generation cycle, Radiat. Res., 32 (1967) 327-346. 21 D. Bootsma and R. M. Humphrey, The progression of mammalian cells through the division cycle following ultraviolet radiation, Mutat. Res., 5 (1968) 289-298. 22 M. Domon and R. M. Rauth, Ultraviolet-light irradiation of mouse L ceils: effects on DNA synthesis and progression through the cell cycle, Radiut. Res., 3.5 (1968) 350-368. 23 M. Domon and R. M. Rauth, Ultraviolet-light irradiation of mouse L cells: effects on cells in the DNA synthesis phase, Radiat. Res., 40 (1969) 414-429. 24 R. H. Rothman and R. B. Setlow, An action spectrum for ceII kihmg and pyrimidine dimer formation in Chinese hamster V79 cells, Photochem. Photobid., 29 (1979) 57-61. 25 F. Ziiizer, J. Kiefer and S. Rase, Inactivation and mutation induction to 6-thioguanine resistance in V79 Chinese hamster fibroblasts by simulated sunlight, Photo&em. Photobiol., 47 (1988) 399-404. 26 P. V. Hariharan and P. A. Cerutti, Formation of products of the 5,6-dihydroxydihydrothymine type by ultraviolet light in HeLa cells, Biochemistry, 16 (1977) 2791-2795. 27 B. S. Rosenstein and J. M. Ducore, Induction of DNA strand breaks in normal human fibroblasts exposed to monochromatic ultraviolet and visible wavelengths in the 240-546 run range, Photo&m. Photobiol., 38 (1983) 51-55. 28 J. G. Peak, M. J. Peak, R. S. Sikorski and C. A. Jones, Induction of DNA-protein crosslinks in human cells by ultraviolet and visible radiations: action spectrum, Photo&em. Photobid., 41 (1985) 295-302.
Photobiol.
B: Biol., 9 (1991)
171-179
171
Changes in cell cycle distribution of V79 Chinese hamster fibroblasts after irradiation at different wavelengths F. Z61zert, B. Heil and J. Kiefer Strahlenzentrum ok Justus-Liebig-Universitfit 6300 G&&n (F.R. G.]
Gi@en,
Leihgesterner
Weg 217,
(Received August 28, 1990; accepted September 10, 1990)
Keywords. UV effects, flow cytometry, DNA damage, pyrimidine dimers.
Abstract Changes in celI cycle distribution of V79 Chinese hamster fibroblasts were investigated at different wavelengths between 254 and 313 run. The fluences applied led to surviving fractions of 0.61. In all cases, the S fraction was temporarily increased within 8-12 h after irradiation, whereas the G1 fraction was decreased. The maximum deviations from the initial values did not signifkantly depend on the wavelength.
1. Introduction During the last few years the wavelength dependences of many different UV radiation effects have been investigated. In bacteria, for instance, action spectra are known for membrane damage [ 1, 21 and the destruction of different transport systems [3-61 as well as for growth delay [ 7, 81. Research with eukaryotes at the same time has been largely restricted to inactivation and mutation induction. There are reports on membrane damage and growth delay in yeast [9] and the inhibition of DNA synthesis in frog cells [lo], but no comparable data for mammalian cells have been published. Thus the present study of cell cycle disturbances in Chinese hamster fibroblasts may be of particular interest. 2. Materials and methods 2.1. Culture cond&kms Chinese hamster fibroblasts V79A received from M. Fox, Manchester, were used for this study. Cultures were grown at 37 “C in Dulbecco’s Modified +Author to whom correspondence should be addressed at: Institut fiir Medizinische Strahlenbiologie, Universit&tskhnikumEssen, Hufelandstrai3e 55, 4300 Essen, F.R.G.
loll-1344/91/$3.50
Q Elsevier Sequoia/Printed in The Netherlands
172
Eagle Medium with 4500 mg I-’ glucose without sodium pyruvate (Gibco, Paisley, U.K.) supplemented with 5% foetal calf serum (Gibco). IV-[2-Hydroxyethyllpiperazine-IV’-[ 2-ethane-sulphonic acid] (HEPES) buffer (1 M; 1 ml 1-l; Seromed, Berlin, F.R.G.), Penicillin (0.06 g 1-l) and streptomycin (0.05 g 1-l) were added to the medium. Cells were subcultured every 3-4 days and regularly checked for mycoplasm. For the experiments, cells from a confhrent culture were plated at a density of lo5 ml-’ on several culture dishes (diameter, 9 cm) containing 10 ml medium and incubated overnight. Before irradiation the medium was withdrawn, traces were washed away with about 10 ml phosphate-buffered saline (PBS) (8 g NaCI, 2 g KCl, 1.5 g Na&IP04, 0.2 g KHpPOl per litre) and 10 ml PBS was added per dish. The cells were then irradiated as described below, after which the buffer was again replaced by 10 ml medium. After incubation for different times, the cells were trypsinized, centrifuged (lOOg, 5 ruin) and fixed in 70% ethanol. 2.2. F7ow fq_mmf?t?y For the staining procedure the cells were again centrifuged (2OOg, 5 mm) and resuspended in 2 ml pepsin solution (0.5 g pepsin (1000 U g-l, Serva), 5.5 ml 1 N HCI and 100 ml distilled water, pH 1.8). After 5 min, tris buffer (12 g tris-(hydroxymethyl)aminomethane, 6 g NaCl, 84 ml 1 N HCl and 1000 ml distilled water, pH 7.5) was added to stop the enzyme reaction. The cells were centrifuged for a third time (2OOg, 5 min), left with 6-8 drops of ribonuclease solution (0.1 g ribonuclease (50 U mg- ‘, Boehringer) in 100 ml distilled water) for 5 min and then resuspended in 4 ml ethidiumbromide-mithramycin solution (1 mg ethidiumbromide, 2.5 mg mithramycin, 305 mg MgClz.6H20 in 100 ml tris buffer). Measurements were performed with a flow cytometer (ICP 22, Phywe). 2.3. Radiation sources A 10 W low pressure mercury lamp (HNS 12, Osram) for 254 run and a 1000 W mercury arc lamp (1000 mercury (xenon), Oriel) equipped with a prism monochromator (designed by Meyer, 1966) for wavelengths between 265 and 313 m-nwere used. The setting of the monochromator was checked with the aid of a spectrophotometer (PMQ 3, Zeiss). The bandwidth (full width at half-maximum) of the radiation was between 2.5 and 4.0 run (see Table 1). As the culture dishes had to be irradiated in a horizontal position, the beam from the monochromator was deflected by a mirror. The resulting radiation field was somewhat inhomogeneous; therefore, the culture dishes were acentrically rotated to ensure as uniform an energy distribution as possible. Instead of the normal plastic lid a quartz cover was used for the culture dishes. The fluence rates were determined separately for each experiment by potassium ferrioxalate actinometry [ 111 (see Table 1). 3. Results The measurements yielded histograms showing the number of cells at different DNA contents. There were normally two clearly distinguishable
173 TABLE
1
Parameters
of irradiation
Wavelength
Bandwidth
Fluence
(nm)
(nm)
(w mm2)
Fluence (J ms2)
254 265 273 283 293 303 313
2.7 3.0 3.3 3.7 3.9 3.1
0.41 0.27 0.12 0.13 0.52 0.45 1.66
5.7 4.5 4.5 5.2 17.1 323.0 6940.0
rate
applied
maxima which could be attributed to the G1 and Gz fractions. For a quantitative evaluation of the histograms, certain assumptions had to be made about the S fraction. We chose two methods of analysis: graphical and computational. In the first case, the S fraction was represented by a rectangle extending from the middle of the G1 peak to the middle of the G2 peak; the areas left and right of the rectangle were taken for the Gr and Ga fractions respectively [ 121. In the second case, the S fraction was approximated by a broadened polynomial and the G1 and Gz fractions by gaussian curves; the sum of these three functions was fitted to the actual distribution by a least-squares procedure 1131. Several studies have shown that the latter method leads to rather accurate results for exponentially growing cultures, as judged by comparison with autoradiographical data [ 141. However, a UV irradiated culture is certainly not growing exponentially and the computational fit was rather unsatisfactory for some of our histograms. We could have added another gaussian curve with variable position to the S fraction [ 151, but that would have greatly increased the computation time. Moreover, it seems that the conclusions drawn do not depend on the specific method of histogram evaluation. The data from the computational analysis are given here; the graphical method leads to very similar results. Changes in the cell cycle distribution were investigated after irradiation at seven different wavelengths which had been used in earlier studies of inactivation (loss of colony forming ability) [ 161. The fluences applied led to surviving fractions of 0.61, i.e. the mean number of “lethal hits” per cell was 0.5. In some cases, the values in Table 1 do not agree with those previously published, since later experiments required certain corrections. However, substantial differences only occurred at 303 nm (323 instead of 165 J rne2) and 313 run (6940 instead of 4170 J mb2). This was due to a reduction in stray light as a consequence of recoating the monochromator mirrors. Samples were taken at 4 h intervals up to 24 h after irradiation. Figure 1 shows a typical series of cell cycle distributions obtained in this way. Each experiment was carried out two to four times, and for each cell cycle
0
100
50
ct!ArwL
150
MJMBER
BOO u b
600
400 200
0 0
Yl
100
150
CHAFNELW-lBER
,000
I
1
Fig. 1. Changes in cell cycle distribution after irradiation at 254 nm. Typical series of histograms obtained at different time intervals. Calculated distributions for G1, S and Gz phases together with the sum of all three functions (-).
175
distribution two histograms were recorded. The G1, S and G2 fractions and their difterences from the initial values were determined. Figure 2 and Table 2 summarize the data obtained. All quantities given are means and the standard errors were obtained from interexperimental variation. Regardless of the wavelength applied, similar changes in cell cycle distribution were observed. After irradiation, the fraction of cells was temporarily increased in the early S phase (4-8 h after irradiation), in the middle S phase (8-12 h after irradiation) and in the late S phase (12-16 h after irradiation). On the whole, the S fraction reached a maximum after 8-12 h, while the G, fraction reached a minimum at the same time. These changes appeared somewhat earlier after short wavelength than after long wavelength irradiation. However, the maximal deviations from the initial values were essentially the same in all cases. When the cells were not irradiated, but otherwise treated in the same manner as described above, no significant changes in cell cycle distribution occurred. The experimental conditions in the absence of irradiation obviously had no effect on cell proliferation. 4. Discussion Changes in cell cycle distribution after UV irradiation at 254 run have been investigated by Imray et al. [ 171 in human lymphoblastoid cells and by KiriIlova et al. [ 18, 191 in V79 Chinese hamster fibroblasts. Both studies support earlier evidence from work with synchronized cultures [20-231 that, in normal cells, only the S phase is delayed, whereas the G1 and G2 phases are not affected. However, in certain repair deficient strains, the passage from G1 to S is also inhibited. The latter effect seems to be related to a low ability of the cells to seal gaps which occur during excision repair or replication of a damaged template (human cells: XP variant; Chinese hamster cells: CHS 2). Our results are in good agreement with those of Imray et al. and Kirillova et al. and further corroborate their conclusions, albeit on different grounds. We were primarily interested in the wavelength dependence of cell cycle disturbances. It has been suggested earlier that, between 254 and 313 nm, the inactivation of Chinese hamster cells correlates with the formation of pyrimidine dimers [ 241. In a more detailed analysis, however, such a correlation was only found for the final slope of the survival curves, whereas the shoulder of the survival curves seemed to be more pronounced at longer wavelengths than at shorter wavelengths [ 16, 251. For the experiments described here, we used fluences which reduced the surviving fraction to 0.61, i.e. almost identical to the shoulder width of the survival curves. Had we sought to induce the same number of dimers in each case, we would have applied 50% smaller fluences at 303 and 313 run. Indeed, a number of earlier experiments were carried out in this way. Since we did not have a suitable computer program at our disposal, the
176
I-
I
0.
4
12
.3
TIME
0
4
8 TIME
16
20
IL.-I
Ih
12 , h
16
20
2L
-7--
__,
A 0
1.
8
0
&
8
12 TIME I h
4
8
IIME
2. Changes in cell cycle distribution after irradiation at 254313 G1 (El), S (0) and Gz (A) fractions. Fig.
20
24
16
20
21
.--A TIME I
0
J 16
A2
12 I h
16
20
24
ruu. Mean values for
177 TABLE
2
Overall effect of irradiation Wavelength 0-w
254 265 273 283 293 303 313 “Sum of deviations
on the cell cycle
Maximal deviation from initial value
Sum of deviations from initial value”
G, (96)
s (%I
G
-21.7k5.5 - 19.3 + 2.6 -20.1*6.6 -15.5k5.6 -21.5k3.5 -33.2*2.9 - 18.8 + 3.3
25.0 f 7.0 29.5 +2.0 21.Ok7.0 16.1 f5.1 19.0 f 6.8 21.6k2.3 17.3 f 8.3
-37.4* 14.2 - 34.8 f 8.2 -70.2k31.7 -41.6& 13.9 - 40.9 & 9.8 -49.6& 13.6 -41.0* 10.0
at 4, 8, 12,
(%I
s (46) 38.3 f 12.8 42.4+8.6 62.7536.7 41.0& 15.6 30.0 f 19.6 40.8 f 10.8 32.1 f 17.5
16 and 20 h.
data were analysed by graphical means only. It is clear, however, that the shift in the G1 and S fractions appeared at an earlier time after irradiation and the maximum deviation was reduced by a factor of about two compared with the results shown in Fig. 2 and Table 2. Thus it is not self-evident that we should observe similar changes in cell cycle distribution when fluences leading to the same survival level are chosen. Loss of colony forming ability is a comparatively late test parameter determined 7 days after irradiation, whereas the effects described here occur within the first 24 h. Nevertheless, there seems to be a correlation between the two end points, suggesting that changes in cell cycle distribution do not just depend on the number of pyrimidine dimers induced, but also on the ability of the cells to recover from sublethal damage. As mentioned above, Imray et al. and Kirillova et al. interpreted their results in much the same way. DNA lesions other than pyrimidine dimers are thought to play a role in the region above 300 run. The relative frequencies of glycols, strand breaks and DNA-protein crosslinks increase with wavelength [26-281. If these types of damage contribute to the effects on the cell cycle, we should not observe similar distributions with comparatively large numbers of dimers at 303 and 313 run, as was the case here, but just the opposite. Rosenstein [lo] reported a coincidence of the action spectra for inhibition of DNA synthesis and formation of endonuclease sensitive sites in frog cells. However, the photoreactive sectors seemed to be slightly reduced at wavelengths above 300 nm, pointing to a role for non-dimer damage. Since the cells used by Rosenstein were deficient in excision repair, the situation may be quite different from ours or, alternatively, inhibition of DNA synthesis may not be the only reason for cell cycle disturbances.
178
Acknowledgments
The cytofluorometrical measurements were performed in the laboratory of D. Kraushaar, Urological Clinic of the University of GiefSen, whose help is gratefully acknowledged. We thank B. Barth for excellent and reliable technical assistance and Dr. T. V. Kirillova for valuable comments on the manuscript. The investigations were supported by the “Bundesministerium fiir Forschung und Technologie” of the F.R.G. through the “Gesellschaft fiir Strahlen- und Umweltforschung” (KBF 49). References 1 L. R. Kelland, S. H. Moss and D. J. G. Davies, An action spectrum for ultraviolet radiation induced membrane damage in Escherichia coli K-12, Photo&em. Photobiol., 37 (1983)
307-312. 2 L. R. Kelland, S. H. Moss and D. J. G. Davies, Leakage of asRbf after ultraviolet irradiation of Eschmichia coli K-12, Photochem. Photobiol., 39 (1984) 329-336. 3 F. T. Robb, J. H. Hauman and M. J. Peak, Similar spectra for the inactivation by monochromatic light of two distinct leucine transport systems of Escherichia coli, Photo&em. Photobiol., 27 (1978) 465-469. 4 J. M. Ascenzi and J. Jagger, Ultraviolet action spectrum (238-405 nm) for inhibition of glycine uptake in E. coli, Photo&em, Photobiol., 30 (1979) 661-666. 5 F. T. Robb and M. J. Peak, Inactivation of the lactose permease of Escherichiu coli by monochromatic ultraviolet light, Photo&em, Photobtil., 30 (1979) 379-383. 6 R. C. Sharma and J. Jagger, Ultraviolet (254-405 run) action spectrum and kinetic studies of alanine uptake in Escherichia coli B/r, Photo&em. Photobiol., 33 (1981) 173-177. 7 J. Jagger, W. C. Wise and R. S. Stafford, Delay in growth and division induced by near ultraviolet radiation in Eschmichia coli B and its role in photoprotection and liquid holding recovery, Photochem. Photobiol., 3 (1964) 11-24. 8 H. E. Kubitschek and M. J. Peak, Action spectrum for growth delay induced by near ultraviolet light ln E. Coli B/r K, Photo&em. Photobiol., 31 (1980) 55-58. 9 J. Kiefer, M. Schall and A. Al-Talibi, Physiological responses of yeast cells to DV of different wavelengths, in R. C. Worrest (ed.), Stratospheric Ozone Reduction, Solar Ultraviolet Radiation and Plant Life, Springer, Berlin, 1986, pp. 151-159. 10 B. S. Rosenstem, Inhibition of semiconservative DNA synthesis in ICR 2A frog cells exposed to monochromatic UV wavelengths (252-313 nm) and photoreactivating light, Radiat. Res., 90 (1982) 509-517. 11 C. G. Hatchard and C. A. Parker, A new sensitive chemical actionometer. II. Potassium ferrioxalate as a standard chemical actionometer, Proc. R. Sot. London, Ser. A, 235 (1956) 518-536. 12 H. Baisch, W. Giihde and W. A. Linden, Analysis of PCP-data to determine the fraction of cells in the various phases of the cell cycle, Radiat. Environ. Biophys., I2 (1975) 3139. 13 P. N. Dean and J. H. Jett, Mathematical analysis of DNA distributions derived from flow microfluorometry, J. Cell. Biol., 60 (1974) 523-527. 14 J. W. Gray, P. N. Dean and M. L. Mendelsohn, Quantitative cell cycle analysis, in M. R. Melamed, P. F. Mullaney and M. L. Mendelsohn (eds.), Flow Cytometry and Sorting, Wiley, New York, 1979, pp. 383-407. 15 M. Fox, A model for the computer analysis of synchronous DNA distributions obtained by flow cytometry, Cytometry, I (1980) 71-77. 16 F. Ziilzer and J. Kiefer, Wavelength dependence of inactivation and mutation induction to 6-thioguanine resistance in V79 Chinese hamster ilbroblasts, Photochem. Photobiol., 40 (1984) 49-53.
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