- Email: [email protected]
16 Kinetics of the DNA-platinum interaction
1048
Biochimie.
1. R o s e n h e r g , B., V a n C a m p , L. & K r i g a s , T. U965) N a ture, 295, 698-69~. 2. M a c q u e t , J. P. a B u t o u r , J. L. (1978) E n r . Y. Binchem., 83, 375-387. 3. B u t o u r , J. L. & M a e q u e t , J. P. (1977) Ear. J Binchem., 78, 455-463. 4. P a s e o e , J. M., R o b e r t s , J. J~ & W o o d w a r d , J. (1974) in <(Recent Results in Cancer Research. Platin u m Coordination Complexes in Cancer Chemotherapy ~>, 48, 108-110. 5. T h i e l ~ D. & C r u s c h l b a u e r , W. (19699 Biopolymers, 8, 3 6 1 - 3 7 8 .
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6. T h ' e l e , D., G u s c h l b a u e r , W . a F a v r e , A. (1972) Biochim. biophys. Acta, 272, 22-26. 7. H o r a c e k , P. ~ I ) r o b n i k , J. (1971) Biochim. biophys. Aria, 254, 341-347. 8. Millard, ~ . M., M.aequet, J. P. & T h ~ o p h a n i d e s , T. (19"t5) Biochim. biophys. Acta, 402, 166-170. 9. M a c q n e t , J. P. ~ T h e o p h a n i d e s , T. (1975) Bioinor 9, Chem., 5, 59-66.
16 Kinetics of the DNA-platinum interaction. J e a n - L u e BUTOUI~ a n d J e a n - P i e r r e MACQUET, Laboratoire de Pharmacologic et de Toxicologic Fondameatales du CNRS, 205 route de Narbonne, 31~978 Toulouse Cedex, France. R e c e n t l y [1] we h a v e r e p o r t e d t h e u s e o f f l u o r e s c e n c e s p e e t r o p h o t o m e t c y in d e t e r m i n i n g t h e p e r t t t r b a t i o n s o f t h e DNA s e c o a d a r y s t r u c t u r e u p o n p l a t i n u m f i x a t i o n a n d a l s o i n p l a t i n u m d e t e r m i n a t i o n of t h e D N A - P l a t i n u m c o m p l e x e s . T h i s t e c h n i q u e is n o w u s e d f o r s t u d y i n g t h e k i n e t i c s of t h e i n t e r a c t i o n b e t w e e n a s e r i e s of p l a t i n u m c o m p o u n d s a n d DNA. T h e m e t h o d c a n o n l y be a p p l i e d to t h e p l a t i n u m c o m p o u n d s W h i c h d e s t a b i l i z e t h e DNA s e c o n d a r y s t r u c t u r e . C o m p o u n d s s u c h as [Pt(NH~)3ClJC1, [ P t ( d i e n ) C l ] C 1 a n d t~'ansPt(NH:0..Cl_o(under r e s t r i c t i v e c o n d i t i o n s ) c a n n o t be s t u d i e d s i n c e t h e y a r e b o u n d to DNA v i a o n l y o n e DNAP t b o n d a n d c o n s e q u e n t l y do n o t a l t e r t h e s t a c k i n g of t h e DNA b a s e s . T h i s i n t e r e s t i n g f e a t u r e ~ ' i l l be d i s c u s s e d i n a n o t h e r c o m m u n i c a t i o n [2] i n r e l a t i o n w i t h t h e c y t o t o x i c p r o p e r t i e s of [Pt(dien)C1]C1 on c u l t u r e d L1210 cells. T ~ e p l a t i n u m c o m p o u n d s u s e d it) t ~ i s s t u d y a r e p r e s e n t e d in t a b l e I. T h e r e a c t i o n s xvere r u n a t 37 ° + O,I'~C in 10-2 M NaC10~, 10-a M NaC1 (10 m l ) a n d t h e p l a t i n u m c o m p o u n d s i n t r o d u c e d c o r r e s p o n d e d to rb = 0.10, 0.20 a n d 0.40. At zero t i m e , p l a t i n u m s o l u t i o n s veere a d d e d to TABLE I. Platinura compounds K~[PtGI4] K[Pt(NH~)CI~] eis-Pt(NHa)~CI 2 cis-Pt( NH~)._,I~ t is-Pt(en)Cl~ lrans-Pt(NH~)~CI. 2 eis-[Pt(NHj).~(H.~O)~I(NOI). ~ cis-[Pt(en)(H~O).2l(NO~).2 trans-[Pt(NHa)2(H~O)~](NO~).,"
BIOCHIMIE, I978, 60, n ° 9.
tl/~ (hours) r~ = 0,~0 8 6.7 3 9 3 3 ~. 5 2,5 < 0 1 ~ (| l O. 5
s a l m o n s p e r m I)NA (0.25 m g / m l ) . A l i q u o t s (0A0 m l ) were t a k e n a t d i f f e r e n t t i m e s a n d a d d e d to 2.4 m~ o f e t h i d i u m b r o m i d e (EtdBr, 40 ~tg/ml) in 0.4 M KNO~. Fluorescence measurements are expressed by the ratio Is/In w h e r e It = f l u o r e s c e n c e i n t e n s i t y of t h e D N A - P t E t d B r c o m p l e x m i n u s f l u o r e s c e n c e i n t e n s i t y of p u r e E t d B r a n d I,, ---- f l u o r e s c e n c e i n t e n s i t y of t h e DNAE t d B r c o m p l e x m i n u s f l u o r e s c e n c e i n t e n s i t y of p u r e E t d B r . It is i m p o r t a n t to m e n t i o n t h a t t h e u s e of NaC1 or KC1 s o l u t i o n s a t a c o n c e n t r a t i o n g r e a t e r t h a n 10-,3 M i n h i b i t t h e D N A - P l a t i n u m i n t e r a c t i o n . F o r i n s t a n c e in t h e ease of cis-Pt(NHj)oCL_, t h e r e is a 50 p e r c e n t i n h i b i t i o n of t h e i n t e r a c t i o n in 50-2 M NaGS a n d m o r e t h a n 90 p e r c e n t i n h i b i t i o n in 10-1 M NAG1. T h i s c l e a r l y d e m o n s t r a t e s t h a t l h e r e a c t i n g s p e c i e s o n DNA is n o t t h e c h l o r i n e s p e c i e s b u t a n a q u o species. If a n o t h e r s a l t like KNO~, NaNny, NaClO~ or Na.SO, is u s e d , no sig~filqcant effect o n t h e D N A - P ~ a t i a u m i n t e r a c f i o ~ c o u l d be d e t e c t e d . Moreover, t h e d i a q u o s p e c i e s of t h e p l a t i n u m c o m p o u n d s r e a c t v e r y q u i c k l y o n DNA (table I). A s i g n i f i c a n t d i f f e r e n c e is e n c o u n t e r e d b e t w e e n t h e cis- a n d trans- d i a q u o species, ~whieh c o u l d reflect t h e k i n e t i c d i f f e r e n c e b e t w e e n c h e l a t i o n (cisb i d e n t a t e ) a n d trans-bidentate p l a t i n u m b i n d i n g . F o r t h e e h l o r o species, t h e k i n e t i c s s e e m s to be g o v e r n e d b y h y d r o l y s i s [3]. 1. B u t o u r , J. L. & M a c q u e t , J. P. (1977) Ear. J. Binchem., 78, 455-463 ; (1978) Anal. Biochem., 89, 22-30. 2. B u t o u r , J. L., M a c q u e t , J. P. & P a o l e t t i , C. ( c o m m u n i c a t i o n n ° 25). 3. T u c k e r , M. A., Colvin, C. B. & M a r t i n , D. S. (1964) Inorg. Chem., 3, ~373.
17
Mass spectrometry study of DNA.cis-Pt(NH~)2CI~ complexes. Christophe JANKOV¢SKI,Ddpartement de Chimie, Unioersit8 de Moncton, Moncton, Nouoeau-Brunsrwielc, E1A 3E9, Canada. J e a n - P i e r r e MAcQuET a n d J e a u - L u e B u T o u n , Laboratoire de Pharmacologic et de Toxicologic Fondamentales du CNRS, 205 route de Narbonne, 31078 Toulouse Cedex, France. T h e c o m b i n a t i o n of P y r o l y s i s a n d M a s s S p e c t r o m e t r y (Py-MS) w a s u s e d to a n a l y s e a s e r i e s o f D N A . cis-Pt(NH~)~C12 c o m p l e x e s . C h a r n o e k a n d Lop [1] a n d W i e b e r s [2] h a v e s u c c e s s f u l l y s u b m i t t e d DNA a n d p o l y d e o x y m b o n u c l e o t i d e s to m a s s s p e c t r o m e t r y ~vithout pretreatment such as derivatization, chemical or enzymatic hydrolysis. The two fundamental papers b y W i e b e r ' s g r o u p [3, 4] c l e a r l y d e m o n s t r a t e t h a t t h i s t e c h n i q u e h a s b e c o m e a n i m p o r t a n t tool i n b o t h det e c t i o n a n d i d e n t i f i c a t i o n o f n u c l e o t i d e s . T h e DNA fragmentation under pyrolytic and electron impact c o n d i t i o n s p r o c e e d s v i a t w o s t e p s : 1. c l e a v a g e o f t h e p h o s p h o d i e s t e r b o n d s a ~ d 2. c l e a v a g e of t h e n u c l e o side, g i v i n g i o n p r o d u c t s 'which c a n be u s e d a s d i a g n o s t i c i o n s f o r b a s e f r a g m e n t s [4]. T~ds ~ast f r a g m e n t a t i o n 'was v e r y 'well s t u d i e d f o r d i f f e r e n t u n d e r i v a t i zed n u c l e o s i d e s [5]. T h e i d e n t i f i c a t i o n of b a s e f r a g m e n t s is c e r t a i n l y t h e e a s i e s t t e c h n i q u e of s t r u c t u r e determination mainly because preliminary degradat i o n is n o t r e q u i r e d a n d t h e q u a n t i t y of c o m p o u n d
Biochimie.
1. R o s e n h e r g , B., V a n C a m p , L. & K r i g a s , T. U965) N a ture, 295, 698-69~. 2. M a c q u e t , J. P. a B u t o u r , J. L. (1978) E n r . Y. Binchem., 83, 375-387. 3. B u t o u r , J. L. & M a e q u e t , J. P. (1977) Ear. J Binchem., 78, 455-463. 4. P a s e o e , J. M., R o b e r t s , J. J~ & W o o d w a r d , J. (1974) in <(Recent Results in Cancer Research. Platin u m Coordination Complexes in Cancer Chemotherapy ~>, 48, 108-110. 5. T h i e l ~ D. & C r u s c h l b a u e r , W. (19699 Biopolymers, 8, 3 6 1 - 3 7 8 .
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.
6. T h ' e l e , D., G u s c h l b a u e r , W . a F a v r e , A. (1972) Biochim. biophys. Acta, 272, 22-26. 7. H o r a c e k , P. ~ I ) r o b n i k , J. (1971) Biochim. biophys. Aria, 254, 341-347. 8. Millard, ~ . M., M.aequet, J. P. & T h ~ o p h a n i d e s , T. (19"t5) Biochim. biophys. Acta, 402, 166-170. 9. M a c q n e t , J. P. ~ T h e o p h a n i d e s , T. (1975) Bioinor 9, Chem., 5, 59-66.
16 Kinetics of the DNA-platinum interaction. J e a n - L u e BUTOUI~ a n d J e a n - P i e r r e MACQUET, Laboratoire de Pharmacologic et de Toxicologic Fondameatales du CNRS, 205 route de Narbonne, 31~978 Toulouse Cedex, France. R e c e n t l y [1] we h a v e r e p o r t e d t h e u s e o f f l u o r e s c e n c e s p e e t r o p h o t o m e t c y in d e t e r m i n i n g t h e p e r t t t r b a t i o n s o f t h e DNA s e c o a d a r y s t r u c t u r e u p o n p l a t i n u m f i x a t i o n a n d a l s o i n p l a t i n u m d e t e r m i n a t i o n of t h e D N A - P l a t i n u m c o m p l e x e s . T h i s t e c h n i q u e is n o w u s e d f o r s t u d y i n g t h e k i n e t i c s of t h e i n t e r a c t i o n b e t w e e n a s e r i e s of p l a t i n u m c o m p o u n d s a n d DNA. T h e m e t h o d c a n o n l y be a p p l i e d to t h e p l a t i n u m c o m p o u n d s W h i c h d e s t a b i l i z e t h e DNA s e c o n d a r y s t r u c t u r e . C o m p o u n d s s u c h as [Pt(NH~)3ClJC1, [ P t ( d i e n ) C l ] C 1 a n d t~'ansPt(NH:0..Cl_o(under r e s t r i c t i v e c o n d i t i o n s ) c a n n o t be s t u d i e d s i n c e t h e y a r e b o u n d to DNA v i a o n l y o n e DNAP t b o n d a n d c o n s e q u e n t l y do n o t a l t e r t h e s t a c k i n g of t h e DNA b a s e s . T h i s i n t e r e s t i n g f e a t u r e ~ ' i l l be d i s c u s s e d i n a n o t h e r c o m m u n i c a t i o n [2] i n r e l a t i o n w i t h t h e c y t o t o x i c p r o p e r t i e s of [Pt(dien)C1]C1 on c u l t u r e d L1210 cells. T ~ e p l a t i n u m c o m p o u n d s u s e d it) t ~ i s s t u d y a r e p r e s e n t e d in t a b l e I. T h e r e a c t i o n s xvere r u n a t 37 ° + O,I'~C in 10-2 M NaC10~, 10-a M NaC1 (10 m l ) a n d t h e p l a t i n u m c o m p o u n d s i n t r o d u c e d c o r r e s p o n d e d to rb = 0.10, 0.20 a n d 0.40. At zero t i m e , p l a t i n u m s o l u t i o n s veere a d d e d to TABLE I. Platinura compounds K~[PtGI4] K[Pt(NH~)CI~] eis-Pt(NHa)~CI 2 cis-Pt( NH~)._,I~ t is-Pt(en)Cl~ lrans-Pt(NH~)~CI. 2 eis-[Pt(NHj).~(H.~O)~I(NOI). ~ cis-[Pt(en)(H~O).2l(NO~).2 trans-[Pt(NHa)2(H~O)~](NO~).,"
BIOCHIMIE, I978, 60, n ° 9.
tl/~ (hours) r~ = 0,~0 8 6.7 3 9 3 3 ~. 5 2,5 < 0 1 ~ (| l O. 5
s a l m o n s p e r m I)NA (0.25 m g / m l ) . A l i q u o t s (0A0 m l ) were t a k e n a t d i f f e r e n t t i m e s a n d a d d e d to 2.4 m~ o f e t h i d i u m b r o m i d e (EtdBr, 40 ~tg/ml) in 0.4 M KNO~. Fluorescence measurements are expressed by the ratio Is/In w h e r e It = f l u o r e s c e n c e i n t e n s i t y of t h e D N A - P t E t d B r c o m p l e x m i n u s f l u o r e s c e n c e i n t e n s i t y of p u r e E t d B r a n d I,, ---- f l u o r e s c e n c e i n t e n s i t y of t h e DNAE t d B r c o m p l e x m i n u s f l u o r e s c e n c e i n t e n s i t y of p u r e E t d B r . It is i m p o r t a n t to m e n t i o n t h a t t h e u s e of NaC1 or KC1 s o l u t i o n s a t a c o n c e n t r a t i o n g r e a t e r t h a n 10-,3 M i n h i b i t t h e D N A - P l a t i n u m i n t e r a c t i o n . F o r i n s t a n c e in t h e ease of cis-Pt(NHj)oCL_, t h e r e is a 50 p e r c e n t i n h i b i t i o n of t h e i n t e r a c t i o n in 50-2 M NaGS a n d m o r e t h a n 90 p e r c e n t i n h i b i t i o n in 10-1 M NAG1. T h i s c l e a r l y d e m o n s t r a t e s t h a t l h e r e a c t i n g s p e c i e s o n DNA is n o t t h e c h l o r i n e s p e c i e s b u t a n a q u o species. If a n o t h e r s a l t like KNO~, NaNny, NaClO~ or Na.SO, is u s e d , no sig~filqcant effect o n t h e D N A - P ~ a t i a u m i n t e r a c f i o ~ c o u l d be d e t e c t e d . Moreover, t h e d i a q u o s p e c i e s of t h e p l a t i n u m c o m p o u n d s r e a c t v e r y q u i c k l y o n DNA (table I). A s i g n i f i c a n t d i f f e r e n c e is e n c o u n t e r e d b e t w e e n t h e cis- a n d trans- d i a q u o species, ~whieh c o u l d reflect t h e k i n e t i c d i f f e r e n c e b e t w e e n c h e l a t i o n (cisb i d e n t a t e ) a n d trans-bidentate p l a t i n u m b i n d i n g . F o r t h e e h l o r o species, t h e k i n e t i c s s e e m s to be g o v e r n e d b y h y d r o l y s i s [3]. 1. B u t o u r , J. L. & M a c q u e t , J. P. (1977) Ear. J. Binchem., 78, 455-463 ; (1978) Anal. Biochem., 89, 22-30. 2. B u t o u r , J. L., M a c q u e t , J. P. & P a o l e t t i , C. ( c o m m u n i c a t i o n n ° 25). 3. T u c k e r , M. A., Colvin, C. B. & M a r t i n , D. S. (1964) Inorg. Chem., 3, ~373.
17
Mass spectrometry study of DNA.cis-Pt(NH~)2CI~ complexes. Christophe JANKOV¢SKI,Ddpartement de Chimie, Unioersit8 de Moncton, Moncton, Nouoeau-Brunsrwielc, E1A 3E9, Canada. J e a n - P i e r r e MAcQuET a n d J e a u - L u e B u T o u n , Laboratoire de Pharmacologic et de Toxicologic Fondamentales du CNRS, 205 route de Narbonne, 31078 Toulouse Cedex, France. T h e c o m b i n a t i o n of P y r o l y s i s a n d M a s s S p e c t r o m e t r y (Py-MS) w a s u s e d to a n a l y s e a s e r i e s o f D N A . cis-Pt(NH~)~C12 c o m p l e x e s . C h a r n o e k a n d Lop [1] a n d W i e b e r s [2] h a v e s u c c e s s f u l l y s u b m i t t e d DNA a n d p o l y d e o x y m b o n u c l e o t i d e s to m a s s s p e c t r o m e t r y ~vithout pretreatment such as derivatization, chemical or enzymatic hydrolysis. The two fundamental papers b y W i e b e r ' s g r o u p [3, 4] c l e a r l y d e m o n s t r a t e t h a t t h i s t e c h n i q u e h a s b e c o m e a n i m p o r t a n t tool i n b o t h det e c t i o n a n d i d e n t i f i c a t i o n o f n u c l e o t i d e s . T h e DNA fragmentation under pyrolytic and electron impact c o n d i t i o n s p r o c e e d s v i a t w o s t e p s : 1. c l e a v a g e o f t h e p h o s p h o d i e s t e r b o n d s a ~ d 2. c l e a v a g e of t h e n u c l e o side, g i v i n g i o n p r o d u c t s 'which c a n be u s e d a s d i a g n o s t i c i o n s f o r b a s e f r a g m e n t s [4]. T~ds ~ast f r a g m e n t a t i o n 'was v e r y 'well s t u d i e d f o r d i f f e r e n t u n d e r i v a t i zed n u c l e o s i d e s [5]. T h e i d e n t i f i c a t i o n of b a s e f r a g m e n t s is c e r t a i n l y t h e e a s i e s t t e c h n i q u e of s t r u c t u r e determination mainly because preliminary degradat i o n is n o t r e q u i r e d a n d t h e q u a n t i t y of c o m p o u n d