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Effects of platinum antitumor agents and pyrimidine dimers on the in vitro replication of T7 DNA
Chem.-Biol. Interactions, 23 (1978) 267--271 © Elsevier/North-Holland Scientific Publishers Ltd.
267
E F F E C T S OF PLATINUM ANTITUMOR AGENTS AND PYRIMIDINE DIMERS ON THE IN V I T R O REPLICATION O F T7 D N A
NEIL P. JOHNSON, JAMES D. HOESCHELE*, NANCY B. KUEMMERLE, WARREN E. MASKER and RONALD O. RAHN
Biology Division and *Health and Safety Research Division, Oak Ridge National Laboratory, Oak Ridge, Tenn. 37830 (U.S.A.) (Received June 13th, 1978) (Accepted July 12th, 1978)
Introduction Numerous chemical and physical agents with pathological or medicinal effects interact with the bases o f DNA and impair normal DNA replication. Although the ability of many agents to alter DNA template activity towards non-semiconservative replication with DNA polymerases has been investigated [ 1 ] , there appears to be little knowledge of the relative effectiveness per lesion with which various agents inhibit replication. The purpose of this paper is to quantitate the extent of DNA replication on templates of T7 DNA with k n o w n amounts of pyrimidine dimers or covalently bound platinum complexes attached. The platinum c o m p o u n d s studied are cis-dichlorodiammine platinum(II) (c/s-DDP), which is an effective antitumor agent, and trans-dichlorodiammine platinum(II) (trans-DDP), which has no antitumor activity [ 2 ] . We found that c/s-DDP and pyrimidine dimers inhibited DNA replication to the same extent per lesion (63% inhibition for 3 × 10 -4 lesions/nucleotide phosphate) and the trans-DDP was 5-fold less inhibitory. Materials and Methods We have used an in vitro T7 DNA replication system [3] which copies exogenous T7 D N A by a mechanism that closely mimics in vivo DNA replication. The D N A products o f this reaction are biologically active [4,5 and unpublished d a t a ] , and inhibition o f in vitro DNA synthesis in this system can presumably result from interference with any of the steps involved in the m o v e m e n t o f the replication fork. Hence, the in vitro system resembles the in vivo situation closely, and the mechanisms of inhibition in vivo and in vitro are likely to be similar. In vitro replication has the advantage that the DNA template may be manipulated exogenously, producing a known number o f lesions on each DNA molecule before presenting the DNA to the N.P.J. was an American Cancer Society Postdoctoral Fellow. Abbreviations: cis-DDP, c/s-dichlorodiammine platinurn(II); trans-DDP, trans-dichlorodiammine platinurn(II).
268 replication machinery for copying. However, the extract used for in vitro replication also contains some endogenous DNA whose template activity must be subtracted from the total and whose products may interfere with a clear demonstration of the size of the DNA synthesized from the damaged template. Preparation of the T7 DNA template. Solutions containing 2.25 × 10 -4 M highly purified T7 DNA [6], from 8.3 × 10-7--4.5 × 10 -6 M ~gsmptlabeled DDP (Hoeschele, J.D., unpublished results), and 5 mM NaC104 were reacted at 37°C. Two 0.043-ml aliquots were removed at various times up to 3 h; one portion was frozen immediately for the replication experiment, while the other was used to determine R b from the acid-insoluble radioactivity by a filter paper assay [7]. For R b = 1 0 - s , 19Smpt radioactivity was only 20 cpm over background for this a m o u n t of DNA; R b < 1 0 -s was estimated from extrapolation of a plot of Rb versus total platinum added for identical reaction conditions. DNA synthesis for a particular R b was independent o f the initial DDP/DNA ratio. In separate experiments T7 DNA was irradiated and the number of pyrimidine dimers was determined as reported previously [8]. Replication o f the T7 DNA. Escherichia coli strain W3110 (thyA su-) was grown with shaking at 37°C in L broth until a cell density of about 1 × 109 cells/ml was reached. The exponentially growing cells were infected at a multiplicity of infection of 5 with bacteriophage T7 carrying amber mutations in gene 3 (am 29) and gene 6 (am 147) [9], and the infection was allowed to proceed for 14.5 min before the cells were chilled and collected. An extract was prepared from these phage-infected cells as previously described [3,4]. 20-pl portions of this extract were added to reaction mixtures (0.1 ml final vol.) which contained final concentrations of 30 mM Tris--HC1 (pH 7.5); 20 mM MgC12 ; 10 mM mercaptoethanol; 0.3 mM each ATP, GTP, CTP, UTP, TTP, dGTP, dCTP; and [32P]dATP at 0.75 cpm/pmol (New England Nuclear, Boston, Mass.). The reaction mixtures also included 3 nmol of T7 DNA template prepared as above. After 10 or 20 min incubation at 30°C the reaction mixture were chilled and new synthesized DNA was determined from the acid-insoluble radioactivity by a filter apper assay [7]. Contributions of *gsrnpt to the radioactivity were subtracted by use of the appropriate control. The rate of synthesis,~ 175 pmol/min, was linear with time for more than 20 min in these experiments.
Results and Discussion The rate of DNA replication was determined for T7 DNA templates containing a known number of either pyrimidine dimers or bound 19smpt-labeled cis- or trans-DDP. The variation in the percent of DNA synthesized as a function of the n u m b e r of lesions per nucleotide phosphate (R b) is shown in Fig. 1. At an Rb of 3 × 10 -4 , cis-DDP reduces the rate of synthesis by 63%; trans-DDP is approx. 5 times less effective. The presence of unreacted cis- or trans-DDP during DNA synthesis does n o t alter the replication rate (data not shown), hence we conclude that these compounds primarily affect the
269
I00-
~ n
¢~ 80i,i -r Z >- 60O9 ,~ ~, E~
o~ 40-
20-
' 1C~-~
'1(~-~
' 1C~-~ ~
" 1C~-~ s=
MOLAR RATIO OF DNA LESIONS (R b) Fig. 1. DNA synthesized in vitro from T7 DNA templates with various numbers o f lesions. The amount o f DNA synthesized without exogenous DNA was subtracted from the total DNA synthesized. Results are presented as percent DNA synthesized relative to a sample with no lesions. Each data point is the average o f percent DNA synthesized at 10 and 20
min. (?) cis-DPP NH~p-~--/CI(~) trans-DPP N~/~-~...Cl (.) pyrimidinedimerspy/Xpy. NH~" "CI CI NH~ DNA template rather than enzymes important to replication. The limited results presented for UV-irradiated DNA clearly indicate that pyrimidine dimers act as replication inhibitors with an efficiency similar to that for c/s- but n o t trans-DDP. In vivo [10], and by inference in vitro, replication of a T7 molecule begins at a single site and proceeds bidirectionally. The presence of a lesion can inhibit the progress of the replication fork, but does n o t (at least for pyrimidine dimers [8] ) stimulate repair synthesis in the in vitro system. On the basis o f the results presented here, it is n o t possible to state whether the replication machinery can eventually bypass the lesion and continue to replicate the same strand or w h e t h e r it returns to the initiation site and begins to synthesize a new strand. Either of these two events would undoubtedly slow down the rate of replication. Assuming random distribution of lesions, we conclude that the replication or possibly repair machinery is better able to cope with the trans, than with the cis-DDP lesion. This may in part reflect the difference in the binding of these two isomers. The cis, but n o t the trans, isomer is able to chelate adjacent bases on the same DNA strans [2]. Pyrimidine dimers also represent a lesion in which adjacent bases on the same strand are coupled [11], therefore the similar inhibitory effect
270 caused by pyrimidine dimers and the cis-DDP lesion m a y reflect the similarity in the structure of the lesion. It is important to remember that the physical and biological nature of the DNA being synthesized from modified templates is n o t known. For example, even when the rate of synthesis is reduced b y only 10%, the DNA molecules being synthesized may n o t be viable T7 genomes. Hence biological inactivation due to replication failure m a y occur at much lower Rb values. Shooter et al. [ 1 2 ] , for example, have reported that cis-DDP inactivates bacteriophage T7 in vivo by 63% at a b o u t Rb = 5 × 10 -s , a level at which only 10% of the in vitro DNA synthesis is inhibited; trans-DDP inactivates phage 7 times less effectively than cis, consistent with less effective inhibition of replication that we observed in vitro. It is o f interest to compare the results in Fig. 1 with those obtained by Harder and Smith [ 1 3 ] , who studied in vitro DNA synthesis using a singlestranded template and DNA ~ and ~ obtained from cultured human lymphoblastoid cells. Their system is inherently less sensitive to modifications of the DNA, since synthesis can initiate at many priming sites on the template. With this system, inhibition of synthesis by 63% required Rb = 0.05 for cis-DDP and a factor o f 2 more trans-DDP. Clearly, repair-like DNA synthesis by DNA polymerases does n o t approach the levels of sensitivity o f the in vivo situation, although even under these conditions the lower effectiveness of the trans isomer was observed. It is highly speculative at this point to discuss our results on the platinum c o m p o u n d s in the light of the antitumor activity; nevertheless, it is tempting to suggest that the difference between the c/s and trans isomers noticed here may arise from some enzyme recognition (or binding) mechanisms which m a y also operate, at least in part, when antitumor activity is being expressed. Research sponsored b y the Division o f Biomedical and Environmental Research, U.S. Department of Energy under contract W-7405-eng-26 with the Union Carbide Corporation. 1 N.R. Cozzarelli, The mechanism o f action of inhibitors o f DNA synthesis, Ann. Rev. Biochem., 46 (1977) 641. 2 B. Rosenberg, Platinum coordination complexes in cancer chemotherapy, Naturwissenschaften, 60 (1973) 399. 3 O.C. Hinkle and C.C. Richardson, Bacteriophage T7 deoxyribonucleic acid replication in vitro, J. Biol. Chem., 249 (1974) 2974. 4 W.E. Maker and C.C. Richardson, Bacteriophage T7 deoxyribonucleic acid replication in vitro. VI. Synthesis of biologically active T7 DNA, J. Mol. Biol., 100 (1976) 543. 5 N.B. Kuemmerle and W.E. Masker, In vitro packaging of UV-radiation-damaged DNA from bacteriophage T7, J. Virol., 23 (1977) 509. 6 C.C. Richardson, the 5'-terminal nucleotides o f T7 bacteriophage deoxyribonucleic acid, J. Mol. Biol., 15 (1966) 49. 7 F.J. Bollum, Filter paper disk techniques for assaying radioactive molecules, Proc. Nucl. Acid Res., 1 (1966) 296. 8 W.E. Masker, Deoxyribonucleic acid repair in vitro by extracts of Escherichia coli, J. Bacteriol., 129 (1977) 1415.
271 9 F.W. Studier, Bacteriphage T7, Science 176 (1972) 367. 10 D. Dressier, J. Wolfson and M. Magazin, Initiation and reinitiation of DNA synthesis during replication of bacteriophage T7, Proc. Natl. Acad. Sci. U.S.A. 69 (1972) 998. 11 M.H. Patrick and R.O. Rahn, Photochemistry of DNA and polynucleotides" Photoproducts, in: S.Y. Wang (Ed.), Photochemistry and Photobiology of Nucleic Acids, Vol. 2, Academic Press, London, 1976, 35--95. 12 K.V. Shooter, R. Howse, R.K. Merrifield and R.B. Robins, The interaction of platinum II compounds with bacteriophages T7 and R17, Chem.-Biol. Interact., 5 (1972) 289. 13 H.C. Harder and R.G. Smith, Effects of mono- and bifunctional platinum compounds on template activity of alternating copolymeric nucleotides, J. Clin. Hematol. Oncol., 7 (1977) 401.
267
E F F E C T S OF PLATINUM ANTITUMOR AGENTS AND PYRIMIDINE DIMERS ON THE IN V I T R O REPLICATION O F T7 D N A
NEIL P. JOHNSON, JAMES D. HOESCHELE*, NANCY B. KUEMMERLE, WARREN E. MASKER and RONALD O. RAHN
Biology Division and *Health and Safety Research Division, Oak Ridge National Laboratory, Oak Ridge, Tenn. 37830 (U.S.A.) (Received June 13th, 1978) (Accepted July 12th, 1978)
Introduction Numerous chemical and physical agents with pathological or medicinal effects interact with the bases o f DNA and impair normal DNA replication. Although the ability of many agents to alter DNA template activity towards non-semiconservative replication with DNA polymerases has been investigated [ 1 ] , there appears to be little knowledge of the relative effectiveness per lesion with which various agents inhibit replication. The purpose of this paper is to quantitate the extent of DNA replication on templates of T7 DNA with k n o w n amounts of pyrimidine dimers or covalently bound platinum complexes attached. The platinum c o m p o u n d s studied are cis-dichlorodiammine platinum(II) (c/s-DDP), which is an effective antitumor agent, and trans-dichlorodiammine platinum(II) (trans-DDP), which has no antitumor activity [ 2 ] . We found that c/s-DDP and pyrimidine dimers inhibited DNA replication to the same extent per lesion (63% inhibition for 3 × 10 -4 lesions/nucleotide phosphate) and the trans-DDP was 5-fold less inhibitory. Materials and Methods We have used an in vitro T7 DNA replication system [3] which copies exogenous T7 D N A by a mechanism that closely mimics in vivo DNA replication. The D N A products o f this reaction are biologically active [4,5 and unpublished d a t a ] , and inhibition o f in vitro DNA synthesis in this system can presumably result from interference with any of the steps involved in the m o v e m e n t o f the replication fork. Hence, the in vitro system resembles the in vivo situation closely, and the mechanisms of inhibition in vivo and in vitro are likely to be similar. In vitro replication has the advantage that the DNA template may be manipulated exogenously, producing a known number o f lesions on each DNA molecule before presenting the DNA to the N.P.J. was an American Cancer Society Postdoctoral Fellow. Abbreviations: cis-DDP, c/s-dichlorodiammine platinurn(II); trans-DDP, trans-dichlorodiammine platinurn(II).
268 replication machinery for copying. However, the extract used for in vitro replication also contains some endogenous DNA whose template activity must be subtracted from the total and whose products may interfere with a clear demonstration of the size of the DNA synthesized from the damaged template. Preparation of the T7 DNA template. Solutions containing 2.25 × 10 -4 M highly purified T7 DNA [6], from 8.3 × 10-7--4.5 × 10 -6 M ~gsmptlabeled DDP (Hoeschele, J.D., unpublished results), and 5 mM NaC104 were reacted at 37°C. Two 0.043-ml aliquots were removed at various times up to 3 h; one portion was frozen immediately for the replication experiment, while the other was used to determine R b from the acid-insoluble radioactivity by a filter paper assay [7]. For R b = 1 0 - s , 19Smpt radioactivity was only 20 cpm over background for this a m o u n t of DNA; R b < 1 0 -s was estimated from extrapolation of a plot of Rb versus total platinum added for identical reaction conditions. DNA synthesis for a particular R b was independent o f the initial DDP/DNA ratio. In separate experiments T7 DNA was irradiated and the number of pyrimidine dimers was determined as reported previously [8]. Replication o f the T7 DNA. Escherichia coli strain W3110 (thyA su-) was grown with shaking at 37°C in L broth until a cell density of about 1 × 109 cells/ml was reached. The exponentially growing cells were infected at a multiplicity of infection of 5 with bacteriophage T7 carrying amber mutations in gene 3 (am 29) and gene 6 (am 147) [9], and the infection was allowed to proceed for 14.5 min before the cells were chilled and collected. An extract was prepared from these phage-infected cells as previously described [3,4]. 20-pl portions of this extract were added to reaction mixtures (0.1 ml final vol.) which contained final concentrations of 30 mM Tris--HC1 (pH 7.5); 20 mM MgC12 ; 10 mM mercaptoethanol; 0.3 mM each ATP, GTP, CTP, UTP, TTP, dGTP, dCTP; and [32P]dATP at 0.75 cpm/pmol (New England Nuclear, Boston, Mass.). The reaction mixtures also included 3 nmol of T7 DNA template prepared as above. After 10 or 20 min incubation at 30°C the reaction mixture were chilled and new synthesized DNA was determined from the acid-insoluble radioactivity by a filter apper assay [7]. Contributions of *gsrnpt to the radioactivity were subtracted by use of the appropriate control. The rate of synthesis,~ 175 pmol/min, was linear with time for more than 20 min in these experiments.
Results and Discussion The rate of DNA replication was determined for T7 DNA templates containing a known number of either pyrimidine dimers or bound 19smpt-labeled cis- or trans-DDP. The variation in the percent of DNA synthesized as a function of the n u m b e r of lesions per nucleotide phosphate (R b) is shown in Fig. 1. At an Rb of 3 × 10 -4 , cis-DDP reduces the rate of synthesis by 63%; trans-DDP is approx. 5 times less effective. The presence of unreacted cis- or trans-DDP during DNA synthesis does n o t alter the replication rate (data not shown), hence we conclude that these compounds primarily affect the
269
I00-
~ n
¢~ 80i,i -r Z >- 60O9 ,~ ~, E~
o~ 40-
20-
' 1C~-~
'1(~-~
' 1C~-~ ~
" 1C~-~ s=
MOLAR RATIO OF DNA LESIONS (R b) Fig. 1. DNA synthesized in vitro from T7 DNA templates with various numbers o f lesions. The amount o f DNA synthesized without exogenous DNA was subtracted from the total DNA synthesized. Results are presented as percent DNA synthesized relative to a sample with no lesions. Each data point is the average o f percent DNA synthesized at 10 and 20
min. (?) cis-DPP NH~p-~--/CI(~) trans-DPP N~/~-~...Cl (.) pyrimidinedimerspy/Xpy. NH~" "CI CI NH~ DNA template rather than enzymes important to replication. The limited results presented for UV-irradiated DNA clearly indicate that pyrimidine dimers act as replication inhibitors with an efficiency similar to that for c/s- but n o t trans-DDP. In vivo [10], and by inference in vitro, replication of a T7 molecule begins at a single site and proceeds bidirectionally. The presence of a lesion can inhibit the progress of the replication fork, but does n o t (at least for pyrimidine dimers [8] ) stimulate repair synthesis in the in vitro system. On the basis o f the results presented here, it is n o t possible to state whether the replication machinery can eventually bypass the lesion and continue to replicate the same strand or w h e t h e r it returns to the initiation site and begins to synthesize a new strand. Either of these two events would undoubtedly slow down the rate of replication. Assuming random distribution of lesions, we conclude that the replication or possibly repair machinery is better able to cope with the trans, than with the cis-DDP lesion. This may in part reflect the difference in the binding of these two isomers. The cis, but n o t the trans, isomer is able to chelate adjacent bases on the same DNA strans [2]. Pyrimidine dimers also represent a lesion in which adjacent bases on the same strand are coupled [11], therefore the similar inhibitory effect
270 caused by pyrimidine dimers and the cis-DDP lesion m a y reflect the similarity in the structure of the lesion. It is important to remember that the physical and biological nature of the DNA being synthesized from modified templates is n o t known. For example, even when the rate of synthesis is reduced b y only 10%, the DNA molecules being synthesized may n o t be viable T7 genomes. Hence biological inactivation due to replication failure m a y occur at much lower Rb values. Shooter et al. [ 1 2 ] , for example, have reported that cis-DDP inactivates bacteriophage T7 in vivo by 63% at a b o u t Rb = 5 × 10 -s , a level at which only 10% of the in vitro DNA synthesis is inhibited; trans-DDP inactivates phage 7 times less effectively than cis, consistent with less effective inhibition of replication that we observed in vitro. It is o f interest to compare the results in Fig. 1 with those obtained by Harder and Smith [ 1 3 ] , who studied in vitro DNA synthesis using a singlestranded template and DNA ~ and ~ obtained from cultured human lymphoblastoid cells. Their system is inherently less sensitive to modifications of the DNA, since synthesis can initiate at many priming sites on the template. With this system, inhibition of synthesis by 63% required Rb = 0.05 for cis-DDP and a factor o f 2 more trans-DDP. Clearly, repair-like DNA synthesis by DNA polymerases does n o t approach the levels of sensitivity o f the in vivo situation, although even under these conditions the lower effectiveness of the trans isomer was observed. It is highly speculative at this point to discuss our results on the platinum c o m p o u n d s in the light of the antitumor activity; nevertheless, it is tempting to suggest that the difference between the c/s and trans isomers noticed here may arise from some enzyme recognition (or binding) mechanisms which m a y also operate, at least in part, when antitumor activity is being expressed. Research sponsored b y the Division o f Biomedical and Environmental Research, U.S. Department of Energy under contract W-7405-eng-26 with the Union Carbide Corporation. 1 N.R. Cozzarelli, The mechanism o f action of inhibitors o f DNA synthesis, Ann. Rev. Biochem., 46 (1977) 641. 2 B. Rosenberg, Platinum coordination complexes in cancer chemotherapy, Naturwissenschaften, 60 (1973) 399. 3 O.C. Hinkle and C.C. Richardson, Bacteriophage T7 deoxyribonucleic acid replication in vitro, J. Biol. Chem., 249 (1974) 2974. 4 W.E. Maker and C.C. Richardson, Bacteriophage T7 deoxyribonucleic acid replication in vitro. VI. Synthesis of biologically active T7 DNA, J. Mol. Biol., 100 (1976) 543. 5 N.B. Kuemmerle and W.E. Masker, In vitro packaging of UV-radiation-damaged DNA from bacteriophage T7, J. Virol., 23 (1977) 509. 6 C.C. Richardson, the 5'-terminal nucleotides o f T7 bacteriophage deoxyribonucleic acid, J. Mol. Biol., 15 (1966) 49. 7 F.J. Bollum, Filter paper disk techniques for assaying radioactive molecules, Proc. Nucl. Acid Res., 1 (1966) 296. 8 W.E. Masker, Deoxyribonucleic acid repair in vitro by extracts of Escherichia coli, J. Bacteriol., 129 (1977) 1415.
271 9 F.W. Studier, Bacteriphage T7, Science 176 (1972) 367. 10 D. Dressier, J. Wolfson and M. Magazin, Initiation and reinitiation of DNA synthesis during replication of bacteriophage T7, Proc. Natl. Acad. Sci. U.S.A. 69 (1972) 998. 11 M.H. Patrick and R.O. Rahn, Photochemistry of DNA and polynucleotides" Photoproducts, in: S.Y. Wang (Ed.), Photochemistry and Photobiology of Nucleic Acids, Vol. 2, Academic Press, London, 1976, 35--95. 12 K.V. Shooter, R. Howse, R.K. Merrifield and R.B. Robins, The interaction of platinum II compounds with bacteriophages T7 and R17, Chem.-Biol. Interact., 5 (1972) 289. 13 H.C. Harder and R.G. Smith, Effects of mono- and bifunctional platinum compounds on template activity of alternating copolymeric nucleotides, J. Clin. Hematol. Oncol., 7 (1977) 401.