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c mice differ in their radiosensitivity
Copynghf 0 1981 by Academic Pre*\. Inc. All rights of reproductmn m any form reserved ool4-48?7/xlillol I I-o7$o?.Mvo
Experimental
DIFFERENTIATED DIFFER HANS
JGBST
Cell Research 136 (1981) 111-l 17
CELLS IN THEIR
FROM
BALB/c
MICE
RADIOSENSITIVITY
RAHMSDGRF,’ HELMUT PONTA, KARL-FRIEDRICH WEIBEZAHN
MARGARETE and PETER
BACHLE, HERRLICH
UDO
MALLICK
SUMMARY Various isolated cells of an inbred mouse strain (BALB/c) differed widely in their sensitivity to gamma irradiation: fibroblasts are five times more resistant than peripheral lymphocytes. Among lymphocytes, T cells are more resistant than B cells. Cell lines derived from the primary cells conserved their radiosensitivity. Cytofluorometric measurements show that the differential reaction of a cell to gamma irradiation can be detected already 2-3 h after the irradiation event. Radiation-sensitive cells are delayed for a longer time in S phase and G2 phase of the cell cycle than radiation-resistant cells. No difference in the capacity of the cells to perform single-strand break repair, double-strand break repair or unscheduled DNA synthesis could yet be detected.
Although the steps of enzymatic DNA repair in eukaryotic cells are still largely unknown, we begin to think of them as regulated functions. Similarly to some of the bacterial enzymes [l-3] eukaryotic repair functions seem inducible [49] and cellcycle dependent [lo]. Some enzymes are possibly involved in cellular processes other than DNA repair, since human genetic deficiencies of DNA repair suffer from disturbances in differentiation [ 111. This led us to examine whether various differentiated cells from a genetically homogeneous multicellular organism differ in the ability to repair DNA. Obvious medical applications of radiation (bone marrow transplant, tumor therapy) suggest such differences between cells, but various other explanations are possible: (i) the primary lesions in the DNA may differ, e.g. because of differences in oxygen tension, endogenous protecting agents, etc.; (ii) 8-811819
rapidly multiplying cells may merely appear radiosensitive because of their turnover upon transient stop of replication. We avoid these drawbacks by isolating cells and comparing their response to gamma irradiation in cell culture. To rule out any influence of the rate of proliferation of primary isolates, we included, in addition, transformed cells of the same origin in our study. The results indicate that cells of different lineages but having the same genetic constitution differ widely in their capacity to repair. MATERIAL
AND
METHODS
Cells All cells were derived from inbred BALB/c mice. Lymphocytes were isolated from the spleens of &IOweek-old female mice and cultured in RPMI 1640 supplemented with 10% fetal calf serum (FCS), ’ To whom
offprint
requests
should
be addressed.
Rahmsdorf
I12
a I Primary cells
et ~1. b)
Cell hnes
y - ray dose [Gray]
Fig. 1. Cells of the same organism differ in radiation sensitivity. (u) Primury cells: Fibroblasts are lung fibroblasts obtained by trypsinization of lung tissue of 3-4-week-old BALB/c mice and subcultured for 5 weeks. B and T cells are from the spleens of the same mice and were differentiated by stimulation with LPS (B cells) respectively conA (T cells). T and B cells received gamma irradiation at the time of mitogenic stimulation, tibroblasts 24 h after passaging. The dose rate was 140 rad/min. DNA synthesis of the cultures was determined by a 2 h [RH]thymidine pulse 48 h
after irradiation. (b) Cell lineA: Fibroblasts are the embryonic fibroblasts BALB/c CL.7 (0-O) and BALB/c 0.5 NG A.1 (W-m). The T-lymphoma cell is S 49.1 (O-O) and the mveloma cells are MPC I1 (X-X) and MPC 21 (+-+)I After irradiation aliquots of the cell cultures were counted every day and growth curves of the surviving cells were constructed. At a time point after irradiation at which all cultures had resumed logarithmic cell growth absolute cell numbers were compared and the survival curves calculated.
2 mM glutamine, 50 PM 2-mercaptoethanol, penicillin (100 units/ml) and streptomycin (100 pg/ml). B cells were activated by the addition of lipopolysaccharide (LPS, 25 pg/ml), T cells by the addition of concanavalin A (conA, 2 &ml). B cells were seoarated from T cells by agg~mmatjon with soybean lectin and subsequent sedimentation f121. T cells were deoleted of B cells by absorbing the B cells to Petri dishes coated with anti-mouse immunoglobulins [l3]. The separation of B and T cells worked efficiently, as tested by the reaction of the cell fractions to LPS (B cells) or conA (T cells) and by direct immunofluorescence. After soybean agglutination the aggregated fraction contained 75-80% immunoglobulinbearing cells, in contrast to the unseparated spleen cell population (around 30% immunoglobulin-bearing cells). Absorbing the B cells specifically to IgC-coated Petri dishes resulted in a non-absorbed cell population containing less than 5 % immunoglobulin-bearing cells which was highly responsive to conA. Lung fibroblasts were obtained by trypsinization of lung tissue and subculturing the outgrowing tibroblasts for 5 weeks in minimum essential medium (MEM) containing Earle’s salts supplemented with IS% FCS and Bykomycin (100 pg/ml). Permanent cell lines, also derived from BALB/c mice. were obtained from the cell distribution center of the Salk Institute for Biological Studies, San Diego, Calif.; BALB/c CL.7 is an embryonic fibroblast [Ii], BALB/c 0.5 NC A.1 was obtained from the former by transformation with nitro-
soguanidine, S 49.1 is a cell line derived from a BALB/c tumor with T cell properties (Thy antigen positive, TL antigen positive, sensitive to corticosteroids, thymidine and phytohemagglutinin [15], MPC I I and MPC 21 were subcultured from mineral oilinduced B cell tumors [16]. They secrete immunoglobulins. All permanent cells were grown in MEMEarle’s supplemented with I5 % FCS and Bykomycin.
E.rp Cell
Res 136 (1981)
@cells. Gamma irradiation was performed with a ““Co gamma source at a dose rate of 1.4 Cy/ min or 35 Gylmin.
Irrudiution
Determination
of radiosensitivity
As the B and T cell-derived cell lines grow in suspension and colony assays in soft agar gave-only low plating efftciencies, survival of all examined cells, including fibroblasts after irradiation was determined by following the growth kinetics of the cells for about IO days. At a time point after irradiation at which all cells resumed logarithmic growth, we compared their cell number.
Surtitwl.
synfhesis. As mitogen-stimulated B and T cells do not grow continuously, we determined their radiosensitivity by measuring their capacity to synthesize DNA at 48 h after gamma irradiation, taking this parameter as a measure of surviving cells. About 2XlO” lymphocytes or ftbroblasts were pulse-labeled for
DNA
Vmrying
radiosensitivity
of d$erentiated
113
cells
2. The different radiosensitivity of B and T cells persists during mitogenic stimulation. The radiosensitivity of B and T lymphocytes was tested as described in fig. I a. Gamma irradiation was given at the time of mitogenic stimulation (A); I6 h (B), 26 h (C); and 48 h (D) thereafter. After irradiation, cells were incubated for 48 h and then tested for DNA synthesis capacity.
Fig.
y-my
dose IGroyl
times up to 2 h with 10 &i [:‘H]thymidine/ml (41 Cilmmol) in growth medium; incorporation was stopped by addition of 5 ml ice-cold PBS to the cells. The cells were pelleted, lysed with 1% sodiumdodecylsulfate (SDS), the DNA precipitated with 5% trichloroacetic acid (TCA) and the radioactivity determined after filtration of the precipitate over GFC filters. Cytojluorometry The distribution of cells in the different phases of the cell cycle was followed in a cytofluorometer after staining of the cellular DNA with EB [ 171. Repair meusurements Single- and double-strand breaks were determined with cells which had been prelabelled for 24+t8 h with 0.05 $Zi [Y]thymidine/ml in growth medium. of total DNA strund breuks. Strand break induction and repair was followed after irradiation of the cells with 9 GY gamma at 3PC at a dose rate of 34 Gylmin. The cells were cooled immediately after irradiation or after they had passed the repair time indicated: by adding IO ml ice-cold PBS (suspension cultures) or bv discarding the medium and adding an ice-cold trypsinfEDTA mix‘iure (0.25 % trypsin, 0.5 mM EDTA) to detach the fibroblasts. The amount of breaks in the DNA was determined after a denaturation-renaturation cvcle bv senaration of single- and double-stranded DNA on hydroxyapatite [18]. About 90% of the breaks induced by gamma irradiation are single-strand breaks [ 191.
Determination
breaks. Double-strand break induction and repair was followed after irradiation of the cells with 50 Gy at a dose rate of 34 Gy/min. Irradiation and repair were performed at 21 or 3PC. The amount of double-strand breaks was determined by a neutral filter elution technique [20]. Double-strund
Unscheduled DNA synthesis CUDS). UDS was determined according to Smith & Hanawalt [2l]. Briefly.
to dense-label the replication forks of DNA, cells were incubated with IO-” M bromodeoxvuridine (BrdUrd) for 2 h. Then about 5 x IO” cells were resuspended in 3 ml of a growth medium containing 3% FCS (no BrdUrd) and irradiated at room temperature with a dose rate of 34 Gylmin. Hydroxyurea (2.5 mM), BrdUrd (lo-” M) and 10 &i [3H]thymidine/ml were added and the incubation continued for 90 min. DNA was prepared by proteinase K digestion in SDS and fractionated according to density in CsCI. The amount of [SH]thymidine incorporated into the light DNA fraction is a measure of UDS performed by the cells.
RESULTS Primary display gammu
AND DISCUSSION
cultures of diflerentiuted different sensitivity to irradiation
cells
We isolated and cultured primary cells from three compartments of an inbred BALB/c mouse: lung fibroblasts, spleen B and T cells. T and B cells were separated by agglutination by soybean lectin respectively by adherence to immunoglobulin-coated Petri dishes and were stimulated by either a B cell (LPS) or a T cell (conA) mitogen. The cells show gross differences in their response to gamma irradiation. The fibroblasts were about 5 times more resistant than the T lymphocytes (fig. 1a). The B cells were twice as sensitive than the T cells. The pronounced radiation sensitivity of lymphocytes was not caused by a radiosensitive step during transformation but Exp
Cell
Res
136 (1981)
114
Ruhmsdorf
8
et al.
12
16
20
lime afterlrmdlotlon I hours1
b
Fig. S. Cell cycle deviations after gamma-irradiation. ((I) Histograms taken at different times after gamma irradiation of BALB/c 0.5 NG A. 1 (fibroblasts), S49.1 (T-lymphoma) and MPC I1 (myeloma); (h) percentage G2+M phase of of cells in (/ej?) S phase and (right) the cell cycle, calculated from similar histograms as displayed in ((I). Cells were irradiated with 6 Gray at 37°C in growth medium at a dose rate of 140 rdd/ min. Similar differences in the reaction of the various cells were obtained when the cells were irradiated at
a dose rate of 3 500 rad/min. This is important because in most of the repair measurements the high dose rate has been employed. At the indicated times control and irradiated cell cultures were harvested and processed for cytofluorometry. The left panel in ((I) displays the histograms of not irradiated logarithmically growing cells. From these it can be calculated that in all three cell lines about 50% of the cells are in S phase of the cell cycle and about 15% are in G2+M phase (h, horizontal bars).
of these cells: At different times after polyclonal activation, the radiosensitivity of lymphocytes did not change substantially, and the difference in radiosensitivity between B and T cells persisted (fig. 2).
responding differentiation lineages: fibroblast lines (BALB/c CL.7, BALB/c 0.5 NG A.l), a T cell line (S 49.1), B cell lines (MPC 11, MPC21) all from BALB/c mice, had similar cell cycle characteristics (fig. 3b), and still responded to gamma irradiation as did their precursor cells. The fibroblast lines were the most resistant and the myeloma cells the most sensitive (fig. 1b). This convinced us that individual cells indeed differed in some step of a complex chain of reactions and we examined then steps of repair accessible to us.
was
an intrinsic
property
The dilferences in radiosensitivity oj primary cells ure conserved in cell lines derived from these cells
The differences were not mediated by differences in cell cycle or proliferation since cell lines which were derived from the corExp Cell
Res 136 (19811
Varying
a 0’
I
I
rtrdiosensitil’it?.
e\”
0
a E
I 100
Timeafter irradiation [mini Fig. 4. Repair of total DNA strand breaks. The cells are the same as in fig. 3. Repair was allowed at 3PC as described in Material and Methods. Each point is the mean value of triplicate determinations.
Crrmmtr-irrtrdiLltedfihroh(rrsts, T und B cells, ure delrryedfbr rwrying periods in S and 62 phuse of cell cycle
Gamma irradiation causes an arrest of the cells in the S phase and the G2 phase [22]. The degree of delay in these two phases was measured by cytofluorometry and corresponds to the survival. The two fibroblast lines tested showed almost no detectable delay in S phase of the cell cycle and recovered completely from G2 delay within 10 h after irradiation (fig. 3). B and T cells, however, were strongly delayed in S phase of the cell cycle. T cells overcame the arrest in G2 only 24 h after gamma irradiation. At this time most of the B cells were still in G2. Thus the delay in G2 paralleled the radiation sensitivity of the cells. The qffect on DNA synthesis IZWSmeasured, in uddition, by thymidine incorpomtion. Gamma irradiation induces a transient
suppression of incorporation of thymidine into DNA, indicating a suppression of DNA synthesis [23]. Although a less reliable parameter than cytofluorometry, the suppression of thymidine incorporation at 1-2 h
0 0
0
8/o x/ z?
1
50-
xx
0 Flbroblost 0 T - lymphomo x Myelomo I 50
115
cells
100
z .c .&z I-
50 -x
of d@krentiated
0 FI broblost o T- lymphomo
0/ 00 0 x II li
x Myelomo I 50
I 100
lime after irmdiotion [min1 Fi
after gamma irradiation was always less severe in fibroblasts than in lymphocytes (results not shown).
Gamma irradiation induces single-strand breaks, double-strand breaks and various base damages in DNA. We tested singlestrand break induction and repair by hydroxyapatite chromatography of DNA, double-strand breaks by a neutral filter elution technique and repair polymerization by determining the amount of unscheduled DNA synthesis. The cells did not differ in any of these parameters. Both, total strand break repair and double-strand break repair were found to be very fast processes, reaching plateau levels within 10 min (at 37’C, and 30 min at 21°C) after irradiation. The cells neither differed in their repair rate nor in the Exl,
Cell
Rrs
136 //98/j
116
Rahmsdorf
et al.
v) .E
1000
y-raydase [Gray] Fig. 6. Unscheduled DNA synthesis induced by gamma irradiation. The cells were BALBlc 0.5 NG A. I (fibroblasts) and S49.1 (T-lymphoma). They were irradiated at room temperature at a dose rate of 35 Gy/min (gamma). Unscheduled DNA synthesis was measured as described in Material and Methods. The amount of radioactivity incorporated into the nonreplicating DNA fraction was corrected for the amount of DNA applied to the CsCl gradient and plotted against the dose given to the cells. Similar results were obtained for the myeloma cell line MPC 11.
amount of breaks which remained nonrejoined (figs 4, 5). Unscheduled DNA synthesis should reflect the excision of modified bases from DNA and the resynthesis of the deleted DNA sequences. Because after gamma irradiation the synthesis of only short stretches of DNA is observed [24], high doses of irradiation had to be applied, in order to induce thymidine incorporation above detection level. The examined cells did not differ in the amount of unscheduled DNA synthesis (fig. 6). CONCLUSION Individual cells of different differentiation pathways differ in radiosensitivity. This may explain their behavior after whole body irradiation. We speculate that the enE.rp Cell
Res
136 llY8/)
zymes involved in generating a certain radiosensitivity are part of the differentiation pathway. Upon transformation to permanent growth, the repair property in the cells examined is apparently retained as is the expression of immunoglobulins in myeloma cells or the synthesis of collagen in fibroblast-derived lines. The experimental conditions permit to conclude that the primary DNA lesions were quantitatively equal in all cells. Also genetically the cells were identical. Thus, the observed differences must originate from different repair behavior. The presently available repair assays do not, however, detect these differences. The different radiosensitivity of the cells with respect to survival or replication was correlated with typical delays in cell cycle kinetics, which thus may in fact serve as fast tests of radiosensitivity (in radiation therapy). But explaining molecular differences between radioresistant and radiosensitive cells must await better understanding of the enzymatic steps involved in gamma-induced DNA repair. We thank Dr Th. Coquerelle for help with the technique of single- and double-strand DNA break determination, and Mr L. Hieber for the measurement and calculation of the cytofluorograms.
REFERENCES 1. McEntee, K, Proc natl acad sci US 74 (1977) 5275. 2. Karran, P, Lindahl, T & Griffin, B, Nature 280 (1979) 76. 3. Kenyon, C J &Walker, G C, Proc natl acad sci US 77 (1980) 2819. 4. Samson, L & Schwartz, J L. Nature 287 (1980) 861. 5. Sarasin, A R & Hanawalt, P C, J mol biol 138 (1980) 299. 6. Friedberg, E C, Moustacci, E. Paul, B R & Ehmann, U K, Possible evidence for inducible repair human cells in culture. Conference on structural pathology in DNA and the biology of ageing. Jahreskonferenz 1979, Zentrallaboratorium fur Mutagenitatspriifung (ed Deutsche Forschungsgemeinschaft). Harald Boldt Verlag (1979).
I.
8.
9. IO. 1 I.
12. 13. 14. 15.
Printed
Mallick, U, Rahmsdorf, H J, Ponta, H, Coquerelle, T, Eife, R & Herrlich. P, Em j cell biol 22 (1980) 102. Mallick, U. Rahmsdorf. H J, Ponta, H & Herrlich, P, Chromosome damage and repair (ed E Seeberg & K Kleppe). Plenum Press, New York. In press. Todd. P, Dalen, H & Schroy. C B, Rad res 69 (1977) 573. Sinclair, W K & Morton, R A. Rad res 29 (1966) 450. Cleaver, J E. Human inherited diseases with altered mechanism for DNA repair and mutagenesis. Excerpta Medica International Congress Series No. 432, Birth Defects. Proc 5th int conf Montreal. Canada, 21-27 Aug. (ed T W Littlefield & J De Grouchy) ElsevierlNorth-Holland Biomedical Press, Amsterdam (1978). Reisner. Y. Ravid, A & Sharon. N, Biochem biophys I-es comm 72 (1976) 1585. Wysocki. L J & Sato, V L. Proc natl acad sci US 75 ( 1978) 2844. Patek, P Q* Collins. J L & Cohn. M. Nature 276 (1978) 510. Ralph. P. Hyman. R, Epstein. R. Nakinz. I &
in Sweden
16. 17. 18. 19. 20. 21. 22. 23. 24.
Cohn. M, Biochem biophys res comm 5.5 (1973) 1085. Potter, M, Physiol rev 52 (1972) 63 I, Lucke-Huhle. C & Dertinger, H, J cancer I3 (1977) 23. Ahnstrom, G & Edvardsson. K. lntj radiat biol26 (1974) 493. Coquerelle. T, Bopp, A. Kessler. B & Hagen, U. Int j radiat biol 24 (1973) 397. Bradley, M 0 & Kohn, K W, Nucleic acids res 7 (1979) 793. Smith. C A & Hanawalt, P C. Biochim biophys acta 432 (1976) 336. Schlag. H. Weibezahn, K F & Lucke-Huhle. C, Int j radiat biol 33 (1978) I. Walters. R A & Hildebrand. C E, Biochem biophys res comm 65 (1975) 265. Lehmann, A R, Molecular biology, biochemistry and biophysics 27 (1978) 312.
Received March 12. 1981 Revised version received May Accepted May 22. 1981
18. 1981
Exp
Cd
Res
136 (1981)
Experimental
DIFFERENTIATED DIFFER HANS
JGBST
Cell Research 136 (1981) 111-l 17
CELLS IN THEIR
FROM
BALB/c
MICE
RADIOSENSITIVITY
RAHMSDGRF,’ HELMUT PONTA, KARL-FRIEDRICH WEIBEZAHN
MARGARETE and PETER
BACHLE, HERRLICH
UDO
MALLICK
SUMMARY Various isolated cells of an inbred mouse strain (BALB/c) differed widely in their sensitivity to gamma irradiation: fibroblasts are five times more resistant than peripheral lymphocytes. Among lymphocytes, T cells are more resistant than B cells. Cell lines derived from the primary cells conserved their radiosensitivity. Cytofluorometric measurements show that the differential reaction of a cell to gamma irradiation can be detected already 2-3 h after the irradiation event. Radiation-sensitive cells are delayed for a longer time in S phase and G2 phase of the cell cycle than radiation-resistant cells. No difference in the capacity of the cells to perform single-strand break repair, double-strand break repair or unscheduled DNA synthesis could yet be detected.
Although the steps of enzymatic DNA repair in eukaryotic cells are still largely unknown, we begin to think of them as regulated functions. Similarly to some of the bacterial enzymes [l-3] eukaryotic repair functions seem inducible [49] and cellcycle dependent [lo]. Some enzymes are possibly involved in cellular processes other than DNA repair, since human genetic deficiencies of DNA repair suffer from disturbances in differentiation [ 111. This led us to examine whether various differentiated cells from a genetically homogeneous multicellular organism differ in the ability to repair DNA. Obvious medical applications of radiation (bone marrow transplant, tumor therapy) suggest such differences between cells, but various other explanations are possible: (i) the primary lesions in the DNA may differ, e.g. because of differences in oxygen tension, endogenous protecting agents, etc.; (ii) 8-811819
rapidly multiplying cells may merely appear radiosensitive because of their turnover upon transient stop of replication. We avoid these drawbacks by isolating cells and comparing their response to gamma irradiation in cell culture. To rule out any influence of the rate of proliferation of primary isolates, we included, in addition, transformed cells of the same origin in our study. The results indicate that cells of different lineages but having the same genetic constitution differ widely in their capacity to repair. MATERIAL
AND
METHODS
Cells All cells were derived from inbred BALB/c mice. Lymphocytes were isolated from the spleens of &IOweek-old female mice and cultured in RPMI 1640 supplemented with 10% fetal calf serum (FCS), ’ To whom
offprint
requests
should
be addressed.
Rahmsdorf
I12
a I Primary cells
et ~1. b)
Cell hnes
y - ray dose [Gray]
Fig. 1. Cells of the same organism differ in radiation sensitivity. (u) Primury cells: Fibroblasts are lung fibroblasts obtained by trypsinization of lung tissue of 3-4-week-old BALB/c mice and subcultured for 5 weeks. B and T cells are from the spleens of the same mice and were differentiated by stimulation with LPS (B cells) respectively conA (T cells). T and B cells received gamma irradiation at the time of mitogenic stimulation, tibroblasts 24 h after passaging. The dose rate was 140 rad/min. DNA synthesis of the cultures was determined by a 2 h [RH]thymidine pulse 48 h
after irradiation. (b) Cell lineA: Fibroblasts are the embryonic fibroblasts BALB/c CL.7 (0-O) and BALB/c 0.5 NG A.1 (W-m). The T-lymphoma cell is S 49.1 (O-O) and the mveloma cells are MPC I1 (X-X) and MPC 21 (+-+)I After irradiation aliquots of the cell cultures were counted every day and growth curves of the surviving cells were constructed. At a time point after irradiation at which all cultures had resumed logarithmic cell growth absolute cell numbers were compared and the survival curves calculated.
2 mM glutamine, 50 PM 2-mercaptoethanol, penicillin (100 units/ml) and streptomycin (100 pg/ml). B cells were activated by the addition of lipopolysaccharide (LPS, 25 pg/ml), T cells by the addition of concanavalin A (conA, 2 &ml). B cells were seoarated from T cells by agg~mmatjon with soybean lectin and subsequent sedimentation f121. T cells were deoleted of B cells by absorbing the B cells to Petri dishes coated with anti-mouse immunoglobulins [l3]. The separation of B and T cells worked efficiently, as tested by the reaction of the cell fractions to LPS (B cells) or conA (T cells) and by direct immunofluorescence. After soybean agglutination the aggregated fraction contained 75-80% immunoglobulinbearing cells, in contrast to the unseparated spleen cell population (around 30% immunoglobulin-bearing cells). Absorbing the B cells specifically to IgC-coated Petri dishes resulted in a non-absorbed cell population containing less than 5 % immunoglobulin-bearing cells which was highly responsive to conA. Lung fibroblasts were obtained by trypsinization of lung tissue and subculturing the outgrowing tibroblasts for 5 weeks in minimum essential medium (MEM) containing Earle’s salts supplemented with IS% FCS and Bykomycin (100 pg/ml). Permanent cell lines, also derived from BALB/c mice. were obtained from the cell distribution center of the Salk Institute for Biological Studies, San Diego, Calif.; BALB/c CL.7 is an embryonic fibroblast [Ii], BALB/c 0.5 NC A.1 was obtained from the former by transformation with nitro-
soguanidine, S 49.1 is a cell line derived from a BALB/c tumor with T cell properties (Thy antigen positive, TL antigen positive, sensitive to corticosteroids, thymidine and phytohemagglutinin [15], MPC I I and MPC 21 were subcultured from mineral oilinduced B cell tumors [16]. They secrete immunoglobulins. All permanent cells were grown in MEMEarle’s supplemented with I5 % FCS and Bykomycin.
E.rp Cell
Res 136 (1981)
@cells. Gamma irradiation was performed with a ““Co gamma source at a dose rate of 1.4 Cy/ min or 35 Gylmin.
Irrudiution
Determination
of radiosensitivity
As the B and T cell-derived cell lines grow in suspension and colony assays in soft agar gave-only low plating efftciencies, survival of all examined cells, including fibroblasts after irradiation was determined by following the growth kinetics of the cells for about IO days. At a time point after irradiation at which all cells resumed logarithmic growth, we compared their cell number.
Surtitwl.
synfhesis. As mitogen-stimulated B and T cells do not grow continuously, we determined their radiosensitivity by measuring their capacity to synthesize DNA at 48 h after gamma irradiation, taking this parameter as a measure of surviving cells. About 2XlO” lymphocytes or ftbroblasts were pulse-labeled for
DNA
Vmrying
radiosensitivity
of d$erentiated
113
cells
2. The different radiosensitivity of B and T cells persists during mitogenic stimulation. The radiosensitivity of B and T lymphocytes was tested as described in fig. I a. Gamma irradiation was given at the time of mitogenic stimulation (A); I6 h (B), 26 h (C); and 48 h (D) thereafter. After irradiation, cells were incubated for 48 h and then tested for DNA synthesis capacity.
Fig.
y-my
dose IGroyl
times up to 2 h with 10 &i [:‘H]thymidine/ml (41 Cilmmol) in growth medium; incorporation was stopped by addition of 5 ml ice-cold PBS to the cells. The cells were pelleted, lysed with 1% sodiumdodecylsulfate (SDS), the DNA precipitated with 5% trichloroacetic acid (TCA) and the radioactivity determined after filtration of the precipitate over GFC filters. Cytojluorometry The distribution of cells in the different phases of the cell cycle was followed in a cytofluorometer after staining of the cellular DNA with EB [ 171. Repair meusurements Single- and double-strand breaks were determined with cells which had been prelabelled for 24+t8 h with 0.05 $Zi [Y]thymidine/ml in growth medium. of total DNA strund breuks. Strand break induction and repair was followed after irradiation of the cells with 9 GY gamma at 3PC at a dose rate of 34 Gylmin. The cells were cooled immediately after irradiation or after they had passed the repair time indicated: by adding IO ml ice-cold PBS (suspension cultures) or bv discarding the medium and adding an ice-cold trypsinfEDTA mix‘iure (0.25 % trypsin, 0.5 mM EDTA) to detach the fibroblasts. The amount of breaks in the DNA was determined after a denaturation-renaturation cvcle bv senaration of single- and double-stranded DNA on hydroxyapatite [18]. About 90% of the breaks induced by gamma irradiation are single-strand breaks [ 191.
Determination
breaks. Double-strand break induction and repair was followed after irradiation of the cells with 50 Gy at a dose rate of 34 Gy/min. Irradiation and repair were performed at 21 or 3PC. The amount of double-strand breaks was determined by a neutral filter elution technique [20]. Double-strund
Unscheduled DNA synthesis CUDS). UDS was determined according to Smith & Hanawalt [2l]. Briefly.
to dense-label the replication forks of DNA, cells were incubated with IO-” M bromodeoxvuridine (BrdUrd) for 2 h. Then about 5 x IO” cells were resuspended in 3 ml of a growth medium containing 3% FCS (no BrdUrd) and irradiated at room temperature with a dose rate of 34 Gylmin. Hydroxyurea (2.5 mM), BrdUrd (lo-” M) and 10 &i [3H]thymidine/ml were added and the incubation continued for 90 min. DNA was prepared by proteinase K digestion in SDS and fractionated according to density in CsCI. The amount of [SH]thymidine incorporated into the light DNA fraction is a measure of UDS performed by the cells.
RESULTS Primary display gammu
AND DISCUSSION
cultures of diflerentiuted different sensitivity to irradiation
cells
We isolated and cultured primary cells from three compartments of an inbred BALB/c mouse: lung fibroblasts, spleen B and T cells. T and B cells were separated by agglutination by soybean lectin respectively by adherence to immunoglobulin-coated Petri dishes and were stimulated by either a B cell (LPS) or a T cell (conA) mitogen. The cells show gross differences in their response to gamma irradiation. The fibroblasts were about 5 times more resistant than the T lymphocytes (fig. 1a). The B cells were twice as sensitive than the T cells. The pronounced radiation sensitivity of lymphocytes was not caused by a radiosensitive step during transformation but Exp
Cell
Res
136 (1981)
114
Ruhmsdorf
8
et al.
12
16
20
lime afterlrmdlotlon I hours1
b
Fig. S. Cell cycle deviations after gamma-irradiation. ((I) Histograms taken at different times after gamma irradiation of BALB/c 0.5 NG A. 1 (fibroblasts), S49.1 (T-lymphoma) and MPC I1 (myeloma); (h) percentage G2+M phase of of cells in (/ej?) S phase and (right) the cell cycle, calculated from similar histograms as displayed in ((I). Cells were irradiated with 6 Gray at 37°C in growth medium at a dose rate of 140 rdd/ min. Similar differences in the reaction of the various cells were obtained when the cells were irradiated at
a dose rate of 3 500 rad/min. This is important because in most of the repair measurements the high dose rate has been employed. At the indicated times control and irradiated cell cultures were harvested and processed for cytofluorometry. The left panel in ((I) displays the histograms of not irradiated logarithmically growing cells. From these it can be calculated that in all three cell lines about 50% of the cells are in S phase of the cell cycle and about 15% are in G2+M phase (h, horizontal bars).
of these cells: At different times after polyclonal activation, the radiosensitivity of lymphocytes did not change substantially, and the difference in radiosensitivity between B and T cells persisted (fig. 2).
responding differentiation lineages: fibroblast lines (BALB/c CL.7, BALB/c 0.5 NG A.l), a T cell line (S 49.1), B cell lines (MPC 11, MPC21) all from BALB/c mice, had similar cell cycle characteristics (fig. 3b), and still responded to gamma irradiation as did their precursor cells. The fibroblast lines were the most resistant and the myeloma cells the most sensitive (fig. 1b). This convinced us that individual cells indeed differed in some step of a complex chain of reactions and we examined then steps of repair accessible to us.
was
an intrinsic
property
The dilferences in radiosensitivity oj primary cells ure conserved in cell lines derived from these cells
The differences were not mediated by differences in cell cycle or proliferation since cell lines which were derived from the corExp Cell
Res 136 (19811
Varying
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Timeafter irradiation [mini Fig. 4. Repair of total DNA strand breaks. The cells are the same as in fig. 3. Repair was allowed at 3PC as described in Material and Methods. Each point is the mean value of triplicate determinations.
Crrmmtr-irrtrdiLltedfihroh(rrsts, T und B cells, ure delrryedfbr rwrying periods in S and 62 phuse of cell cycle
Gamma irradiation causes an arrest of the cells in the S phase and the G2 phase [22]. The degree of delay in these two phases was measured by cytofluorometry and corresponds to the survival. The two fibroblast lines tested showed almost no detectable delay in S phase of the cell cycle and recovered completely from G2 delay within 10 h after irradiation (fig. 3). B and T cells, however, were strongly delayed in S phase of the cell cycle. T cells overcame the arrest in G2 only 24 h after gamma irradiation. At this time most of the B cells were still in G2. Thus the delay in G2 paralleled the radiation sensitivity of the cells. The qffect on DNA synthesis IZWSmeasured, in uddition, by thymidine incorpomtion. Gamma irradiation induces a transient
suppression of incorporation of thymidine into DNA, indicating a suppression of DNA synthesis [23]. Although a less reliable parameter than cytofluorometry, the suppression of thymidine incorporation at 1-2 h
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after gamma irradiation was always less severe in fibroblasts than in lymphocytes (results not shown).
Gamma irradiation induces single-strand breaks, double-strand breaks and various base damages in DNA. We tested singlestrand break induction and repair by hydroxyapatite chromatography of DNA, double-strand breaks by a neutral filter elution technique and repair polymerization by determining the amount of unscheduled DNA synthesis. The cells did not differ in any of these parameters. Both, total strand break repair and double-strand break repair were found to be very fast processes, reaching plateau levels within 10 min (at 37’C, and 30 min at 21°C) after irradiation. The cells neither differed in their repair rate nor in the Exl,
Cell
Rrs
136 //98/j
116
Rahmsdorf
et al.
v) .E
1000
y-raydase [Gray] Fig. 6. Unscheduled DNA synthesis induced by gamma irradiation. The cells were BALBlc 0.5 NG A. I (fibroblasts) and S49.1 (T-lymphoma). They were irradiated at room temperature at a dose rate of 35 Gy/min (gamma). Unscheduled DNA synthesis was measured as described in Material and Methods. The amount of radioactivity incorporated into the nonreplicating DNA fraction was corrected for the amount of DNA applied to the CsCl gradient and plotted against the dose given to the cells. Similar results were obtained for the myeloma cell line MPC 11.
amount of breaks which remained nonrejoined (figs 4, 5). Unscheduled DNA synthesis should reflect the excision of modified bases from DNA and the resynthesis of the deleted DNA sequences. Because after gamma irradiation the synthesis of only short stretches of DNA is observed [24], high doses of irradiation had to be applied, in order to induce thymidine incorporation above detection level. The examined cells did not differ in the amount of unscheduled DNA synthesis (fig. 6). CONCLUSION Individual cells of different differentiation pathways differ in radiosensitivity. This may explain their behavior after whole body irradiation. We speculate that the enE.rp Cell
Res
136 llY8/)
zymes involved in generating a certain radiosensitivity are part of the differentiation pathway. Upon transformation to permanent growth, the repair property in the cells examined is apparently retained as is the expression of immunoglobulins in myeloma cells or the synthesis of collagen in fibroblast-derived lines. The experimental conditions permit to conclude that the primary DNA lesions were quantitatively equal in all cells. Also genetically the cells were identical. Thus, the observed differences must originate from different repair behavior. The presently available repair assays do not, however, detect these differences. The different radiosensitivity of the cells with respect to survival or replication was correlated with typical delays in cell cycle kinetics, which thus may in fact serve as fast tests of radiosensitivity (in radiation therapy). But explaining molecular differences between radioresistant and radiosensitive cells must await better understanding of the enzymatic steps involved in gamma-induced DNA repair. We thank Dr Th. Coquerelle for help with the technique of single- and double-strand DNA break determination, and Mr L. Hieber for the measurement and calculation of the cytofluorograms.
REFERENCES 1. McEntee, K, Proc natl acad sci US 74 (1977) 5275. 2. Karran, P, Lindahl, T & Griffin, B, Nature 280 (1979) 76. 3. Kenyon, C J &Walker, G C, Proc natl acad sci US 77 (1980) 2819. 4. Samson, L & Schwartz, J L. Nature 287 (1980) 861. 5. Sarasin, A R & Hanawalt, P C, J mol biol 138 (1980) 299. 6. Friedberg, E C, Moustacci, E. Paul, B R & Ehmann, U K, Possible evidence for inducible repair human cells in culture. Conference on structural pathology in DNA and the biology of ageing. Jahreskonferenz 1979, Zentrallaboratorium fur Mutagenitatspriifung (ed Deutsche Forschungsgemeinschaft). Harald Boldt Verlag (1979).
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8.
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Mallick, U, Rahmsdorf, H J, Ponta, H, Coquerelle, T, Eife, R & Herrlich. P, Em j cell biol 22 (1980) 102. Mallick, U. Rahmsdorf. H J, Ponta, H & Herrlich, P, Chromosome damage and repair (ed E Seeberg & K Kleppe). Plenum Press, New York. In press. Todd. P, Dalen, H & Schroy. C B, Rad res 69 (1977) 573. Sinclair, W K & Morton, R A. Rad res 29 (1966) 450. Cleaver, J E. Human inherited diseases with altered mechanism for DNA repair and mutagenesis. Excerpta Medica International Congress Series No. 432, Birth Defects. Proc 5th int conf Montreal. Canada, 21-27 Aug. (ed T W Littlefield & J De Grouchy) ElsevierlNorth-Holland Biomedical Press, Amsterdam (1978). Reisner. Y. Ravid, A & Sharon. N, Biochem biophys I-es comm 72 (1976) 1585. Wysocki. L J & Sato, V L. Proc natl acad sci US 75 ( 1978) 2844. Patek, P Q* Collins. J L & Cohn. M. Nature 276 (1978) 510. Ralph. P. Hyman. R, Epstein. R. Nakinz. I &
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Cohn. M, Biochem biophys res comm 5.5 (1973) 1085. Potter, M, Physiol rev 52 (1972) 63 I, Lucke-Huhle. C & Dertinger, H, J cancer I3 (1977) 23. Ahnstrom, G & Edvardsson. K. lntj radiat biol26 (1974) 493. Coquerelle. T, Bopp, A. Kessler. B & Hagen, U. Int j radiat biol 24 (1973) 397. Bradley, M 0 & Kohn, K W, Nucleic acids res 7 (1979) 793. Smith. C A & Hanawalt, P C. Biochim biophys acta 432 (1976) 336. Schlag. H. Weibezahn, K F & Lucke-Huhle. C, Int j radiat biol 33 (1978) I. Walters. R A & Hildebrand. C E, Biochem biophys res comm 65 (1975) 265. Lehmann, A R, Molecular biology, biochemistry and biophysics 27 (1978) 312.
Received March 12. 1981 Revised version received May Accepted May 22. 1981
18. 1981
Exp
Cd
Res
136 (1981)