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Estimation of interstrand DNA cross-linking resulting from mustard gas alkylation of HeLa cells
Chem.-Biol. Interactions
297
Elsevier Publishing Company, Amsterdam Printed in The Netherlands
E S T I M A T I O N OF I N T E R S T R A N D D N A C R O S S - L I N K I N G R E S U L T I N G F R O M M U S T A R D GAS A L K Y L A T I O N OF H e L a CELLS
C. R. BALLa ANDJ. J. ROBERTS b aDepartment of Experimental Pathology and Cancer Research, School of Medicine, Leeds 2, and bChester Beatty Research Institute, Institute of Cancer Research, Royal Cancer Hospital, London (Great Britain)
(Received July 13th, 1971) (Revision accepted September 14th, 1971)
SUMMARY An isopycnic gradient technique is described by which interstrand cross-linking of D N A in mammalian cells resulting from treatment with difunctional alkylating agents can be quantitated.
INTRODUCTION The chromatographic observation 1 of diguaninyl derivativesamongthe hydrolysis products of D N A isolated from E. coli following treatment with [35S]mustard gas led to the hypothesis that difunctional alkylating agents are cytotoxic due to their ability to introduce cross-links into the genome. Such interstrand cross-links were subsequently observed in alkylated bacterial D N A 2'3. By inference it has been implied that the aromatic and other difunctional agents used in cancer chemotherapy act in a similar fashion. The demonstration that mustard gas introduces not only interstrand but also intrastrand cross-links into D N A 4.s has again raised the question as to which alkylation lesion is most significant for the cytotoxic action of these drugs. Measurement of D N A cross-linking is essential to progress in elucidating the mechanism of action of these agents and in understanding the response of D N A repair mechanisms to alkylation by them. A number of methods have been described 5-7 for demonstrating D N A crosslinks in mammalian cells based on analytical ultracentrifugation, methylated-albuminkieselguhr chromatography, thermal denaturation, and biphasic partition methods. This paper describes a method which offers some advantages over previous techniques. Abbreviations: BUdR, 5-bromodeoxyuridine; [3H]TdR, [6-aH]thymidine. Chem.-Biol. Interactions, 4 (1971/72) 297-303
298
C. R. BALL, J. J. ROBERTS
METHODS
Materials [6-3H]Thymidine ([3H]TdR), of specific activity greater than 25 Ci/mmole, was obtained from the Radiochemical Centre, Amersham, Great Britain, and 5-bromodeoxyuridine (BUdR) from the Sigma Chemical Co. Cell cultures H e L a cells were grown in suspension culture in minimal essential Eagle's medium (Grand Island Biological Co., Powder Medium F14) supplemented with 7 ~ foetal calf serum (Biocu|t Laboratories), penicillin and streptomycin. Density labelling procedure In experiments in which no radioactive label was used cells were treated with mustard gas after growing in medium containing BUdR (5/~g/ml) for 20 h. The cells were then centrifuged and D N A subsequently isolated for gradient analysis. In experiments in which a radioactive as well as a density label was introduced into the D N A the following procedure was used. A culture (3 • 105-4 • l0 s cells/ml) was grown in medium containing BUdR (5 pg/ml) for 3 h, the cells recovered by lowspeed centrifugation and resuspended in half the initial volume of fresh BUdR-containing medium. [3H]TdR (1 #Ci/ml) was then added. After a further 3 h the cells were again centrifuged and resuspended in fresh medium containing BUdR (5/zg/ml) at the original cell concentration of 3 • 105-4 • 105 cells/ml. 1 h later the culture was split into a number of smaller cultures for individual treatment with mustard gas in methanol (0.1 ~ , v/v) or with solvent only in the case of controls. 30 min after addition of the drug the cells were centrifuged, washed in isotonic saline, frozen and stored in liquid nitrogen until the following day when the D N A was isolated. DNA isolation and caesium chloride gradients Partially purified D N A was isolated by a modification of the method of KIRBY AND COOK8 as previously described 9. An aliquot (0.5 ml) of a solution of the isolated D N A in 0.15 M sodium chloride-0.015 M sodium citrate was added to 5.6 ml of a 61.9 wt. ~ solution of caesium chloride in sodium phosphate buffer (pH 12.5) to give a density of 1.722. The solution was centrifuged at 40 0O0 rev./min in the l0 ml × l0 ml titanium rotor of an MSE 65 centrifuge at 20 ° for 24 h. The tubes were pierced and 8 drop fractions collected. Each fraction was diluted and its absorbance at 260 nm recorded using an Unicam SP1800 spectrophotometer. An aliquot (1.3 ml) of each diluted sample was added to a Triton X scintillator (10 ml) consisting of toluene (2000 ml), Triton X-100 (1000 g), PPO (16.5 g) and dimethyl POPOP (300 mg), and the radioactivity recorded in a Packard Tri-Carb scintillation counter. RESULTS If HeLa cells are grown in the presence of B U d R for 20 h, which is just less than
Chem.-Biol. Interactions, 4 (1971/72) 297 303
INTERSTRAND D N A CROSS-LINKINGIN HeLa CELLS • :
• CONTROl. : 2 . o g l m l Museard
][~
Gas
299
®
l~'
0"6 O
0-4
0.Z i
FRACTION NUMBER
II0
210"
310
410
Fig. I. Isopycnic caesium chloride gradient of BUdR-density-labelled DNA illustrating the production of an intermediate density band of cross-linked DNA after mustard gas treatment. one cell generation time, then D N A isolated from these cells shows two approximately equal absorbance peaks on an alkaline caesium chloride gradient (Fig. 1) corresponding to the"light" template strand and the "heavy" newly synthesised strand. Treatment of the cells with mustard gas following such density labelling results in a third peak of intermediate density (Fig. 1). The latter peak can only have resulted from covalent chemical bonds between the "light" and "heavy" strands. Although Fig. 1 shows that in principle a method of this type can be used to demonstrate cross-links, it is clearly not suitable for quantitative estimation of the amount of cross-linking. We have therefore designed a radioactive density-labelling technique which overcomes this problem. The labelling regimen results in only the heavy D N A being labelled and thus the cross-linked D N A becomes much more readily observed as a separate peak or a shoulder of radioactivity of intermediate density. The rationale of the labelling procedure, which is described under METHODS, is as follows. The cells are initially grown for 3 h in BUdR-containing medium which ensures that the growing point has moved well away from the "light" D N A before any radioactive label is introduced. The cells are then resuspended in medium containing BUdR and [3H]TdR (this gives identical results to using [3H]BUdR, see ref. 10) so that all DNA synthesised in a further 3-h period is both density labelled and radioactive. The D N A is now suitably labelled for observing cross-linking of the radioactive "heavy" D N A just synthesised to the "old light" DNA. It was thought desirable for a number of reasons to allow the radioactivity-labelled D N A to move away from the growing point before introducing the cross-linking agent. D N A may be initially synthesised in short lengths ~ and has a different secondary structure from " o l d " D N A shortly after synthesis 12. Either of these phenomena could have resulted in lowered sensitivity of the method (see DISCUSSION). To achieve this separation of the labelled D N A from the growing point, the cells were again centrifuged and resuspended in BUdR-containing medium. The presence of BUdR at this stage was essential. In its absence a small but significant peak of radioactivity was observed at intermediate density in control gradients. This was probably due to end addition of unlabelled light D N A to the heavy labelled D N A just synthesised 13 or alternatively Chem.-Biol. Interactions, 4 (1971/72) 297-303
300
C. R. BALL~ J. J. ROBERTS
0"4
8000
CONTROL
7000 0"3
6000
O'Z
4000
SO00
3000 2000
O'l
I000 0
FRACTION NUMBfR I0
20
30
40
Fig. 2A. Caesium chloride gradient o f D N A isolated from cells treated with [3H]TdR and B U d R (see METHODS). 0000
0"4
2~
MUSTARD GAS. 2/uglml
7000 6000
0"3
e.1
5000 (J 4000
]
3000
0.
2000 I000 0
FRACTION NUMBER I0
"
]0
40
Fig. 2B. Caesium chloride gradient of D N A from cells labelled as in Fig. 2A but treated with mustard gas (2 ,ug/ml) before isolation of the D N A . 16 (~
A .~
14 -,.="J 12
\
: = *
-- C O N T R O L : O.Sjug/ml M u = t o r d go= = / j u g / m / M u # t o r d go= • 2 l u g ml M u s t o r d go=
~
'°
• 6 t.J
~ 4 2
F R A C T I O N NUMBER I0
20
29'-')40
Fig. 3. Normalised radioactivity profiles of caesium chloride gradients obtained from cells labelled as in Fig. 2 and treated with a range of mustard gas concentrations. The radioactivity of each fraction is expressed as a percentage of the total radioactivity on that gradient.
due to carry over of [3H]TdR in the nucleotide pool. When B U d R was present a sharp radioactive profile of heavy D N A was obtained (Fig. 2A). Following the final B U d R Chem.-Biol. Interactions, 4 (1971/72) 297-303
INTERSTRAND
D N A CROSS-LINKING 1N HeLa CELLS
301
treatment the cells were treated with various concentrations of mustard gas and the D N A isolated subsequently subjected to isopycnic centrifugation. An intermediate radioactive peak representing cross-linked D N A is then seen (Fig. 2B). Using the method outlined, the relationship between mustard gas concentration and the extent of D N A cross-linking was investigated (Fig. 3). The radioactivity in each fraction of the gradient is plotted in Fig. 3 as the percentage of total radioactivity on the gradient. This normalisation of the curve enables the percentage of crosslinked D N A to be estimated from the area between any one treated curve and the control curve. This can be computed either as the amount of radioactivity lost from the "light" peak or that gained in the intermediate cross-linked peak. The two methods of calculation gave comparable results and these are presented in Fig. 4 as the relationship between amount of cross-linking and mustard gas concentration. 40-
@
Percent D.N.A.
]0 -
cross-linked
ZO-
I0-
o's
,'o
'
1'5
'
Z'O
Concentration (AJg/m/) Fig. 4. T h e percentage cross-linked D N A was estimated from the data o f Fig. 3 in two ways (see RESULTS): × , percentage radioactivity lost from heavy peak; 0 , percentage radioactivity at intermediate density. DISCUSSION
This paper describes a method of quantitating interstrand D N A cross-linking in mammalian cells. In comparison with other methods 6.7, it has the advantage that a large number of samples, up to 10 using MSE or Spinco rotors, can be directly compared simultaneously under conditions where all the D N A samples have been handled identically. Moderately high doses of drug must be used before cross-linking can be readily measured reproducibly. The Do dose of mustard gas for the HeLa cells used is 0.05/zg/ml which is below the limit of sensitivity of the method (Fig. 4). The relative insensitivity is due to shear of D N A molecules during their isolation. The molecular weight of D N A in the nucleus is thought to be at least 2 • 108 daltons and may be as high as 3 • 109 daltons 1~. As the molecular weight of the DNA is decreased by shearing, so will the amount of cross-linking appear to be less since each cross-linked molecule will contribute less radioactivity to the appropriate band on the gradient. Phenol extraction methods of the type used here to isolate the D N A yield D N A with an average molecular weight of Chem.-Biol. Interactions, 4 (1971/72) 297 303
302
C. R. BALL, J. J. ROBERTS
around 2. 10 v daltons. The percentage of cross-linking molecules observed will thus be at least 10-fold less than that actually present in the nucleus. By an admittedly inaccurate extrapolation of the data of Fig. 4, we can see that in the intact nucleus as much as 40 ~o of the D N A may be cross-linked at a dose (0.15 #g/ml) which reduces cell survival to 37 ~ . Clearly, attempts to carry out the method on intact D N A could considerably increase its sensitivity. REiD AND WALKER6 have discussed at length the problems of chain breakage due to shear during isolation of the D N A and also that due to the chemical instability of alkylated DNA. The latter is not a problem in the present paper since all comparisons have been of D N A samples isolated at the same time after alkylation and carried through all the procedures together. The sensitivity of the method described here compares favourably with others in the literature. CHUN e t al. 7 obtained 1 0 ~ cross-linking of Ehrlich ascites D N A only when the level of alkylation reached l alkylation in 20 000 nucleotides or 150 nmoles HN2 per g DNA. From the data of CRATHORN AND ROBERTS15, using the same cell line as used here, we can calculate that 10 ~ cross-linking arises from only 20 nmoles mustard gas per g D N A and this agrees very closely with the data of REID AND WALKER6 for alkylation of L cells by mustard gas. In summary, a method is described for quantitating interstrand cross-links in D N A which is at least as sensitive as any previously described. It should prove useful in elucidating the role of interstrand cross-links relative to other alkylation damage in the cytotoxicity of difunctional alkylating agents. ACKNOWLEDGEMENTS
The authors wish to thank Miss J. GOODBANand Mrs. J. M. PASCOEfor technical assistance. This work was supported in Leeds by the Yorkshire Cancer Research Campaign and by grants to the Chester Beatty Research Institute (Institute of Cancer Research: Royal Cancer Hospital) from the Medical Research Council and the Cancer Research Campaign. REFERENCES 1 2
3 4
5 6
P . D . LAWLEY AND P. BROOKES, T h e action o f alkylating agents on D N A in relation to biological effects o f alkylating agents, Exptl. Cell Res., Suppl. 9 (1963) 512-520. K . W . KOHN, N. H. STEIGBIGEL AND C. L. SPEARS,Cross-linking a n d repair o f D N A in sensitive and resistant strains o f E. coli treated with nitrogen m u s t a r d , Proc. Natl. Acad. Sci. (U.S.), 53 0965) 1154-1161. A. TERAWAKI AND J. GREENBERG, Post-treatment breakage o f mitomycin C-induced cross-links in D N A of E. coli, Biochim. Biophys. Acta, 119 (1966) 540-546. P . O . LAWLEY, J. H. LETHBRIDGE, P. A. EDWARDS AND K. V. SHOOTER, Inactivation of bacteriophage T7 by m o n o - a n d difunctional s u l p h u r m u s t a r d s in relation to cross-linking a n d depurination o f bacteriophage D N A , J. Mol. Biol., 39 (1969) 181-198. I. G. WALKER, I n t r a s t r a n d bifunctional alkylation of D N A in m a m m a l i a n cells treated with m u s t a r d gas, Can. J. Biochem., 49 (1971) 332-336. B . D . REID AND I. G. WALKER, T h e response o f m a m m a l i a n cells to alkylating agents, II. O n the m e c h a n i s m o f removal of s u l p h u r - m u s t a r d - i n d u c e d cross-links, Biochim. Biophys. Acta, 179 (1969) 179-188.
Chem.-Biol. Interactions, 4 (1971/72) 297-303
INTERSTRAND 7
8 9 10
11 12 13 14 15
D N A CROSS-LINKING IN H e L a CELLS
303
E. H. L. CHUN, L. GONZALES,F. S. LEWIS,J. JONES AND R. J. RUTMAN,Differences in in vivo alkylation a n d cross-linking o f nitrogen m u s t a r d sensitive a n d resistant lines o f Lettre-Ehrlich ascites t u m o u r s , Cancer Res., 29 (1969) 1184-1194. K . S . KIRBY AND E. A. COOK,Isolation o f D N A from m a m m a l i a n tissues, Biochem. J., 104 (1967) 254-257. C . R . BALL AND J. J. ROBERTS,D N A repair after m u s t a r d gas alkylation by sensitive and resistant Y o s h i d a s a r c o m a cells in vitro, Chem.-Biol. Interactions, 2 (1970) 321-329. J . J . ROBERTS, J. M. PASCOE, B. A. SMITH AND A. R. CRATHORN, Quantitative aspects o f the repair o f alkylated D N A in cultured m a m m a l i a n cells, 1I. Non-semiconservative D N A synthesis ('repair synthesis') in H e L a a n d Chinese h a m s t e r cells following t r e a t m e n t with alkylating agents, Chem.-Biol. Interactions, 3 (1971 ) 49-68. E . H . HASKELL AND C. I. DAVERN, Pre-fork synthesis: a m o d e l for D N A replication, Proc. Natl. Acad. Sci. (U.S.), 64 (1969) 1065-1071. J . F . HABENER, B. S. BYNUM AND J. SHACK, Destabilised secondary structure o f newly replicated H e L a D N A , J. Mol. Biol., 49 (1970) 157-170. R . B . PAINTER, D. A. JERMANY AND R. E. RASMUSSEN, A m e t h o d to determine the n u m b e r o f D N A replicating units in cultured m a m m a l i a n ceils, J. Mol. Biol., 17 (1966) 47-56. A . R . LEHMANN ANO M. G. ORMEROD, D o u b l e - s t r a n d breaks in the D N A o f a m a m m a l i a n cell after X-irradiation, Biochim. Biophys. Acta, 217 (1970) 268-277. A . R . CRATHORN AND J. J. ROBERTS, M e c h a n i s m o f the cytotoxic action o f alkylating agents in m a m m a l i a n cells a n d evidence for the i e m o v a l o f alkylated groups from deoxyribonucleic acid, Nature, 211 (1966) 150-153.
Chem.-Biol. Interactions, 4 (1971/72) 297-303
297
Elsevier Publishing Company, Amsterdam Printed in The Netherlands
E S T I M A T I O N OF I N T E R S T R A N D D N A C R O S S - L I N K I N G R E S U L T I N G F R O M M U S T A R D GAS A L K Y L A T I O N OF H e L a CELLS
C. R. BALLa ANDJ. J. ROBERTS b aDepartment of Experimental Pathology and Cancer Research, School of Medicine, Leeds 2, and bChester Beatty Research Institute, Institute of Cancer Research, Royal Cancer Hospital, London (Great Britain)
(Received July 13th, 1971) (Revision accepted September 14th, 1971)
SUMMARY An isopycnic gradient technique is described by which interstrand cross-linking of D N A in mammalian cells resulting from treatment with difunctional alkylating agents can be quantitated.
INTRODUCTION The chromatographic observation 1 of diguaninyl derivativesamongthe hydrolysis products of D N A isolated from E. coli following treatment with [35S]mustard gas led to the hypothesis that difunctional alkylating agents are cytotoxic due to their ability to introduce cross-links into the genome. Such interstrand cross-links were subsequently observed in alkylated bacterial D N A 2'3. By inference it has been implied that the aromatic and other difunctional agents used in cancer chemotherapy act in a similar fashion. The demonstration that mustard gas introduces not only interstrand but also intrastrand cross-links into D N A 4.s has again raised the question as to which alkylation lesion is most significant for the cytotoxic action of these drugs. Measurement of D N A cross-linking is essential to progress in elucidating the mechanism of action of these agents and in understanding the response of D N A repair mechanisms to alkylation by them. A number of methods have been described 5-7 for demonstrating D N A crosslinks in mammalian cells based on analytical ultracentrifugation, methylated-albuminkieselguhr chromatography, thermal denaturation, and biphasic partition methods. This paper describes a method which offers some advantages over previous techniques. Abbreviations: BUdR, 5-bromodeoxyuridine; [3H]TdR, [6-aH]thymidine. Chem.-Biol. Interactions, 4 (1971/72) 297-303
298
C. R. BALL, J. J. ROBERTS
METHODS
Materials [6-3H]Thymidine ([3H]TdR), of specific activity greater than 25 Ci/mmole, was obtained from the Radiochemical Centre, Amersham, Great Britain, and 5-bromodeoxyuridine (BUdR) from the Sigma Chemical Co. Cell cultures H e L a cells were grown in suspension culture in minimal essential Eagle's medium (Grand Island Biological Co., Powder Medium F14) supplemented with 7 ~ foetal calf serum (Biocu|t Laboratories), penicillin and streptomycin. Density labelling procedure In experiments in which no radioactive label was used cells were treated with mustard gas after growing in medium containing BUdR (5/~g/ml) for 20 h. The cells were then centrifuged and D N A subsequently isolated for gradient analysis. In experiments in which a radioactive as well as a density label was introduced into the D N A the following procedure was used. A culture (3 • 105-4 • l0 s cells/ml) was grown in medium containing BUdR (5 pg/ml) for 3 h, the cells recovered by lowspeed centrifugation and resuspended in half the initial volume of fresh BUdR-containing medium. [3H]TdR (1 #Ci/ml) was then added. After a further 3 h the cells were again centrifuged and resuspended in fresh medium containing BUdR (5/zg/ml) at the original cell concentration of 3 • 105-4 • 105 cells/ml. 1 h later the culture was split into a number of smaller cultures for individual treatment with mustard gas in methanol (0.1 ~ , v/v) or with solvent only in the case of controls. 30 min after addition of the drug the cells were centrifuged, washed in isotonic saline, frozen and stored in liquid nitrogen until the following day when the D N A was isolated. DNA isolation and caesium chloride gradients Partially purified D N A was isolated by a modification of the method of KIRBY AND COOK8 as previously described 9. An aliquot (0.5 ml) of a solution of the isolated D N A in 0.15 M sodium chloride-0.015 M sodium citrate was added to 5.6 ml of a 61.9 wt. ~ solution of caesium chloride in sodium phosphate buffer (pH 12.5) to give a density of 1.722. The solution was centrifuged at 40 0O0 rev./min in the l0 ml × l0 ml titanium rotor of an MSE 65 centrifuge at 20 ° for 24 h. The tubes were pierced and 8 drop fractions collected. Each fraction was diluted and its absorbance at 260 nm recorded using an Unicam SP1800 spectrophotometer. An aliquot (1.3 ml) of each diluted sample was added to a Triton X scintillator (10 ml) consisting of toluene (2000 ml), Triton X-100 (1000 g), PPO (16.5 g) and dimethyl POPOP (300 mg), and the radioactivity recorded in a Packard Tri-Carb scintillation counter. RESULTS If HeLa cells are grown in the presence of B U d R for 20 h, which is just less than
Chem.-Biol. Interactions, 4 (1971/72) 297 303
INTERSTRAND D N A CROSS-LINKINGIN HeLa CELLS • :
• CONTROl. : 2 . o g l m l Museard
][~
Gas
299
®
l~'
0"6 O
0-4
0.Z i
FRACTION NUMBER
II0
210"
310
410
Fig. I. Isopycnic caesium chloride gradient of BUdR-density-labelled DNA illustrating the production of an intermediate density band of cross-linked DNA after mustard gas treatment. one cell generation time, then D N A isolated from these cells shows two approximately equal absorbance peaks on an alkaline caesium chloride gradient (Fig. 1) corresponding to the"light" template strand and the "heavy" newly synthesised strand. Treatment of the cells with mustard gas following such density labelling results in a third peak of intermediate density (Fig. 1). The latter peak can only have resulted from covalent chemical bonds between the "light" and "heavy" strands. Although Fig. 1 shows that in principle a method of this type can be used to demonstrate cross-links, it is clearly not suitable for quantitative estimation of the amount of cross-linking. We have therefore designed a radioactive density-labelling technique which overcomes this problem. The labelling regimen results in only the heavy D N A being labelled and thus the cross-linked D N A becomes much more readily observed as a separate peak or a shoulder of radioactivity of intermediate density. The rationale of the labelling procedure, which is described under METHODS, is as follows. The cells are initially grown for 3 h in BUdR-containing medium which ensures that the growing point has moved well away from the "light" D N A before any radioactive label is introduced. The cells are then resuspended in medium containing BUdR and [3H]TdR (this gives identical results to using [3H]BUdR, see ref. 10) so that all DNA synthesised in a further 3-h period is both density labelled and radioactive. The D N A is now suitably labelled for observing cross-linking of the radioactive "heavy" D N A just synthesised to the "old light" DNA. It was thought desirable for a number of reasons to allow the radioactivity-labelled D N A to move away from the growing point before introducing the cross-linking agent. D N A may be initially synthesised in short lengths ~ and has a different secondary structure from " o l d " D N A shortly after synthesis 12. Either of these phenomena could have resulted in lowered sensitivity of the method (see DISCUSSION). To achieve this separation of the labelled D N A from the growing point, the cells were again centrifuged and resuspended in BUdR-containing medium. The presence of BUdR at this stage was essential. In its absence a small but significant peak of radioactivity was observed at intermediate density in control gradients. This was probably due to end addition of unlabelled light D N A to the heavy labelled D N A just synthesised 13 or alternatively Chem.-Biol. Interactions, 4 (1971/72) 297-303
300
C. R. BALL~ J. J. ROBERTS
0"4
8000
CONTROL
7000 0"3
6000
O'Z
4000
SO00
3000 2000
O'l
I000 0
FRACTION NUMBfR I0
20
30
40
Fig. 2A. Caesium chloride gradient o f D N A isolated from cells treated with [3H]TdR and B U d R (see METHODS). 0000
0"4
2~
MUSTARD GAS. 2/uglml
7000 6000
0"3
e.1
5000 (J 4000
]
3000
0.
2000 I000 0
FRACTION NUMBER I0
"
]0
40
Fig. 2B. Caesium chloride gradient of D N A from cells labelled as in Fig. 2A but treated with mustard gas (2 ,ug/ml) before isolation of the D N A . 16 (~
A .~
14 -,.="J 12
\
: = *
-- C O N T R O L : O.Sjug/ml M u = t o r d go= = / j u g / m / M u # t o r d go= • 2 l u g ml M u s t o r d go=
~
'°
• 6 t.J
~ 4 2
F R A C T I O N NUMBER I0
20
29'-')40
Fig. 3. Normalised radioactivity profiles of caesium chloride gradients obtained from cells labelled as in Fig. 2 and treated with a range of mustard gas concentrations. The radioactivity of each fraction is expressed as a percentage of the total radioactivity on that gradient.
due to carry over of [3H]TdR in the nucleotide pool. When B U d R was present a sharp radioactive profile of heavy D N A was obtained (Fig. 2A). Following the final B U d R Chem.-Biol. Interactions, 4 (1971/72) 297-303
INTERSTRAND
D N A CROSS-LINKING 1N HeLa CELLS
301
treatment the cells were treated with various concentrations of mustard gas and the D N A isolated subsequently subjected to isopycnic centrifugation. An intermediate radioactive peak representing cross-linked D N A is then seen (Fig. 2B). Using the method outlined, the relationship between mustard gas concentration and the extent of D N A cross-linking was investigated (Fig. 3). The radioactivity in each fraction of the gradient is plotted in Fig. 3 as the percentage of total radioactivity on the gradient. This normalisation of the curve enables the percentage of crosslinked D N A to be estimated from the area between any one treated curve and the control curve. This can be computed either as the amount of radioactivity lost from the "light" peak or that gained in the intermediate cross-linked peak. The two methods of calculation gave comparable results and these are presented in Fig. 4 as the relationship between amount of cross-linking and mustard gas concentration. 40-
@
Percent D.N.A.
]0 -
cross-linked
ZO-
I0-
o's
,'o
'
1'5
'
Z'O
Concentration (AJg/m/) Fig. 4. T h e percentage cross-linked D N A was estimated from the data o f Fig. 3 in two ways (see RESULTS): × , percentage radioactivity lost from heavy peak; 0 , percentage radioactivity at intermediate density. DISCUSSION
This paper describes a method of quantitating interstrand D N A cross-linking in mammalian cells. In comparison with other methods 6.7, it has the advantage that a large number of samples, up to 10 using MSE or Spinco rotors, can be directly compared simultaneously under conditions where all the D N A samples have been handled identically. Moderately high doses of drug must be used before cross-linking can be readily measured reproducibly. The Do dose of mustard gas for the HeLa cells used is 0.05/zg/ml which is below the limit of sensitivity of the method (Fig. 4). The relative insensitivity is due to shear of D N A molecules during their isolation. The molecular weight of D N A in the nucleus is thought to be at least 2 • 108 daltons and may be as high as 3 • 109 daltons 1~. As the molecular weight of the DNA is decreased by shearing, so will the amount of cross-linking appear to be less since each cross-linked molecule will contribute less radioactivity to the appropriate band on the gradient. Phenol extraction methods of the type used here to isolate the D N A yield D N A with an average molecular weight of Chem.-Biol. Interactions, 4 (1971/72) 297 303
302
C. R. BALL, J. J. ROBERTS
around 2. 10 v daltons. The percentage of cross-linking molecules observed will thus be at least 10-fold less than that actually present in the nucleus. By an admittedly inaccurate extrapolation of the data of Fig. 4, we can see that in the intact nucleus as much as 40 ~o of the D N A may be cross-linked at a dose (0.15 #g/ml) which reduces cell survival to 37 ~ . Clearly, attempts to carry out the method on intact D N A could considerably increase its sensitivity. REiD AND WALKER6 have discussed at length the problems of chain breakage due to shear during isolation of the D N A and also that due to the chemical instability of alkylated DNA. The latter is not a problem in the present paper since all comparisons have been of D N A samples isolated at the same time after alkylation and carried through all the procedures together. The sensitivity of the method described here compares favourably with others in the literature. CHUN e t al. 7 obtained 1 0 ~ cross-linking of Ehrlich ascites D N A only when the level of alkylation reached l alkylation in 20 000 nucleotides or 150 nmoles HN2 per g DNA. From the data of CRATHORN AND ROBERTS15, using the same cell line as used here, we can calculate that 10 ~ cross-linking arises from only 20 nmoles mustard gas per g D N A and this agrees very closely with the data of REID AND WALKER6 for alkylation of L cells by mustard gas. In summary, a method is described for quantitating interstrand cross-links in D N A which is at least as sensitive as any previously described. It should prove useful in elucidating the role of interstrand cross-links relative to other alkylation damage in the cytotoxicity of difunctional alkylating agents. ACKNOWLEDGEMENTS
The authors wish to thank Miss J. GOODBANand Mrs. J. M. PASCOEfor technical assistance. This work was supported in Leeds by the Yorkshire Cancer Research Campaign and by grants to the Chester Beatty Research Institute (Institute of Cancer Research: Royal Cancer Hospital) from the Medical Research Council and the Cancer Research Campaign. REFERENCES 1 2
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