Physicochemical and Structural Studies of the In Vitro Interactions

4

P h y s i c o c h e m i c a l a n d S t r u c t u r a l S t u d i e s of the I n V i t r o Interactions between P l a t i n u m ( I I ) Downloaded via UNIV OF MASSACHUSETTS AMHERST on July 10, 2018 at 13:10:53 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

C o m p o u n d s and

DNA

J. P. MACQUET, J. L. BUTOUR, and N. P. JOHNSON

Laboratoire de Pharmacologie et de Toxicologie Fondamentales, CNRS, 205, Route de Narbonne, 31400 Toulouse, France Perturbations of DNA secondary structure and stability induced by c i s - [ P t ( Ν Η ) C l ] (cisDDP), trans-DDP and [Pt(dien)Cl]Cl at low levels of DNA binding (r ) are summarized and preliminary characterization of the corresponding platinum -DNA adducts is presented. For r ≤0.01 both cis -andtrans-DDPform interstrand crosslinks, shorten the DNA and exclude stoichiometric amounts of intercalating agents. However, cis-DDP destabilizes DNA at this r while trans-DDP? stabilizes the polymer and only the cis isomer causes an increase in the circular dichroism of DNA. [Pt(dien)Cl]Cl does not form crosslinks ; it changes neither the length of DNA, the CD spectra nor the intercalation of ethidium bromide. Fixation of [Pt(dien)Cl]Cl stabilizes DNA at r ≤0.01. Two major products of the reaction of cis-DDP with salmon sperm DNA have been isolated and both contain guanine. [Pt(dien)Cl]Cl fixes primarily at Ν of guanine for r < 0.1 ; in addition, fixation at N of adenine for r > 0.1 and at N and Ν of adenine for 0.3 < r < 0.4 was observed. 3

2

2

b

b

b

b

7

7

b

1

7

b

b

Although cis-DDP binds covalently to proteins, RNA and other nucleophiles in the cell (V), it is fixation on DNA which seems to be responsible for the antitumor activity of this compound (2). The toxicity of cis-DDP seems to be associated with an inhibition of DNA synthesis rather than an inhibition of RNA or protein synthesis (3, 4_, 5) ; similarly, mammalian cells (6) and bacteria (_5, _7, 8) deficient in DNA repair are more sensitive to killing by cis-DDP. This argument implies that inhibition of DNA synthesis is responsible for the general cytotoxicity of cisDDP, but it does not explain why tumor cells are killed more read0097-6156/83/0209-0075$07.50/0 © 1983 American Chemical Society Lippard; Platinum, Gold, and Other Metal Chemotherapeutic Agents ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

76

METAL CHEMOTHERAPEUTIC AGENTS

i l y than healthy ones without i n v o k i n g a s p e c i a l s e n s i t i v i t y of tumor c e l l s , such as l a c k of DNA r e p a i r c a p a b i l i t y ( 9 ) . The antitumor a c t i v i t y of a s e r i e s of platinum compounds i s g e n e r a l l y c o r r e l a t e d w i t h t h e i r a b i l i t y to perturb v a r i o u s f u n c t i o n s of DNA such as enhancement of mutagenesis (10, 11, 12), i n h i b i t i o n of DNA s y n t h e s i s (8, 13, 14, 15) and i n d u c t i o n of l y s o g e n i c b a c t e r i a (10, 16). Platinum compounds without antitumor a c t i v i t y (17) such as trans-Wi? and [ P t ( d i e n ) C l ] C l (Figure 1) c o v a l e n t l y b i n d t o DNA in vivo. Several s t u d i e s have compared the b i o l o g i c a l e f f e c t s which r e s u l t when equal amounts of these three platinum compounds are f i x e d on DNA ( t y p i c a l l y rb = 10~4-10""6). Cis-DDP i s 5-10 times more t o x i c toward E. eoli (8) and mammalian c e l l s (J_, JJ_) than trans-DDP. The r e l a t i v e t o x i c i t y i s c o r r e l a t e d w i t h the a b i l i t y of these two isomers to i n h i b i t DNA r e p l i c a t i o n ( c e l l s were g r a f t e d i . p . i n DBA/2 mice on day 0 and animals were t r e a t e d by i . p . i n j e c t i o n w i t h the maximum n o n - t o x i c dose (LDo) of the drug on day 3. For the p e n e t r a t i o n and DNA b i n d i n g experiments, the animals were s a c r i f i e d 2 h a f t e r treatment, the leukemia c e l l s were removed and washed u n t i l no platinum was de t e c t e d i n the supernatant a f t e r c e n t r i f u g a t i o n . The c e l l s were counted and the p l a t i n u m c o v a l e n t l y f i x e d per c e l l was determined by atomic a b s o r p t i o n . DNA was i s o l a t e d from these c e l l s by phenol e x t r a c t i o n and the rb was determined. For the antitumor e x p e r i ments, animals were g r a f t e d w i t h 10^ LI210 c e l l s and observed f o r 30 days. The r e s u l t s are expressed as the mean s u r v i v a l time of t r e a t e d animals (T) d i v i d e d by the mean s u r v i v a l time of nont r e a t e d animals (C). T/C ^ 125 % i s considered as s i g n i f i c a n t antitumor a c t i v i t y .

Compound

LDo (ymol/kg)

T/C (%)

c?is-DDP 30 trans-ΌΏ? 167 [Pt(dien)Cl]Cl 203 [ P t ( e n ) 2 ] C l 2 :> 2,600

200 116 100 98

Pt f i x e d to c e l l s (% treatment dose) 0.4 1.5 0.1 0,02

a

rb

1/30, 000 1/1,200 1/10,000 < 1/200,000

a f t e r 4 washings some f r e e compound i s s t i l l present due to the l a r g e amount i n j e c t e d ; l i m i t of d e t e c t i o n . b

I f the f i x a t i o n of P t ( I I ) compounds on DNA i s r e s p o n s i b l e f o r the v a r i o u s b i o l o g i c a l a c t i v i t i e s of these compounds, then the r e l a t i v e potencies of cis-DDP, trans-ΌΌ? and [ P t ( d i e n ) C l ] C l must be a consequence of t h e i r modes of b i n d i n g on the DNA r a t h e r than the s t o i c h i o m e t r y of f i x a t i o n . The o b j e c t i v e of t h i s paper i s to determine whether or not the d i f f e r e n t b i o l o g i c a l a c t i v i t i e s of these compounds are a s s o c i a t e d w i t h p a r t i c u l a r deformations of DNA. To accomplish t h i s goal we w i l l f i r s t t r y to q u a n t i t a t e the a l t e r a t i o n s of DNA conformation and s t a b i l i t y by these compounds i n vitro at the lowest p o s s i b l e rb- The l i m i t of d e t e c t i o n f o r most of the physicochemical techniques which w i l l be considered i s rb = 0.005. The l e v e l of DNA b i n d i n g which produces most b i o l o g i c a l e f f e c t s i s one or two orders of magnitude l e s s and so we w i l l emphasize r e s u l t s from s t u d i e s w i t h 0.005 < r ^ < 0.05, Since the k i n e t i c s of f i x a t i o n of these compounds are d i f f e r e n t and the amount of p l a t i n u m bound to DNA w i l l change i f s i d e r e a c t i o n s are p o s s i b l e , we w i l l l i m i t our d i s c u s s i o n t o physicochemical s t u d i e s which have measured the platinum on the DNA. G e n e r a l l y p l a t i n u m c o n c e n t r a t i o n has been determined by f l a m e l e s s atomic a b s o r p t i o n or by u s i n g r a d i o a c t i v e 195mpt, both which have a l i m i t of detec t i o n of rb = l-5xlO""6. Secondly, some p r e l i m i n a r y r e s u l t s from our

Lippard; Platinum, Gold, and Other Metal Chemotherapeutic Agents ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

4.

79

Pt(II) Compounds and DNA In Vitro

MACQUET ET AL.

l a b o r a t o r y concerning the i s o l a t i o n and c h a r a c t e r i z a t i o n of the platinum-DNA adducts formed by these compounds w i l l be presented. F i n a l l y * we w i l l d i s c u s s b r i e f l y how the observed s t r u c t u r e s o f the platinum-DNA l e s i o n s may be r e s p o n s i b l e f o r the a l t e r e d s t r u c t u r e and s t a b i l i t y of DNA in vitro. ALTERATIONS OF DNA SECONDARY STRUCTURE AND STABILITY BY P t ( I I ) COMPOUNDS, r < 0.05 b

Before examining, the conformation and s t a b i l i t y of the platinum-DNA complex formed in vitro, i t i s worth b r i e f l y c o n s i d e r i n g the mechanism of the r e a c t i o n between P t ( I I ) chloroammines and DNA. C i s - [ P t ( N H ) 2 C l 2 l does not r e a c t w i t h DNA but the aquated forms, [Pt(NH3)2(H20)Cl] and [ P t ( N H ) ( H 2 0 ) l , bind c o v a l e n t l y to the p o l y n u c l e o t i d e (20) and the o v e r a l l r e a c t i o n l i b e r a t e s 2 CI* (20. 3

+

2 +

3

2

2

[Pt(NH )2Cl ] 3

2

I t +Cl"

-Cl"

[Pt (NH ) ( H 0 ) C l ] 3

2

JJ

-ci

[Pt (NH ) C1 (OH) ]

2

3

+C1~

2

J J +C1

-Cl"

+

-H [Pt ( N H ) (H 0) 1 " ~ ~ = ^ [Pt ( N H ) ( H 0 ) (OH) ] ^ ~ " = Ξ [Pt(NH ) (OH) 1 +

+

3

2

2

2

3

2

2

3

2

2

I f aqueous s o l u t i o n s of ois- o r trans-DDP are allowed t o e q u i l i b r a t e , the k i n e t i c s of the r e a c t i o n s of these aquated species w i t h DNA can be measured. At low r ^ the r e a c t i o n i s p s e u d o - f i r s t order w i t h respect t o platinum c o n c e n t r a t i o n . For cis-DDP, the h a l f - l i f e of the r e a c t i o n of the diaquo species w i t h 10"4 M DNA at 25°C, pH - 5-6 i s 0.8 min and the monoaquo species, c i s - and t r a n s - [ P t ( N H ) ( H 2 0 ) C l ] J have h a l f - l i v e s of 6 h and 2 h r e s p e c t i v e l y . The forms [Pt(NH )2Cl(OH)] and [ P t ( N H ) ( 0 H ) ] do not r e a c t w i t h DNA and c i s - [ P t ( N H ) 2 ( H 2 0 ) ( O H ) ] r e a c t s w i t h the same k i n e t i c s as the diaquo form (20). However, i f f r e s h l y d i s s o l v e d s o l u t i o n s of platinum compounds are added t o the DNA, formation of the monoaquo species i s the r a t e l i m i t i n g step (22). F i g u r e 2 shows the r e a c t i o n s of f r e s h s o l u t i o n s of the three compounds w i t h DNA at 37°C. Under these c o n d i t i o n s the h a l f - l i v e s of the r e a c t i o n s were 3.9, 2.5 and 0.65 h f o r cis-DDP, trans-WV and [ P t ( d i e n ) C l ] C l r e s p e c t i v e l y (19). I t has been proposed that P t ( I I ) compounds b i n d t o DNA i n three d i f f e r e n t ways, c i s - b i d e n t a t e , trarcs-bidentate and monodent a t e , and that the compounds i n F i g u r e 1 are r e p r e s e n t a t i v e of these three c l a s s e s of b i n d i n g (23). We w i l l now consider the e f f e c t o f t h e f i x a t i o n o f t h e s e compounds in vitro, 0.005 Κ rt> < 0.05, on the s t r u c t u r e and the s t a b i l i t y of DNA. 3

2

3

3

2

2

+

3


80


TIME (hours) Figure 2. Kinetics of the reaction between salmon-sperm DNA and cis-DDP (Φ), trms-DDP (M), or [Pt(dien)Cl]Cl (A); U = 0.2. Aliquots of cis- or trans-Z)DP were taken at different times and added to a solution of EtdBr. Fluorescence measurements were performed as previously described (32), and platinum bound to DNA was determined from standard curve of η vs. fluorescence. For [Pt(dien)Cl]Cl, aliquots were taken at different times, and the reaction was stopped by increasing the Cl~ concentration to 1 M. These samples were then passed through Sepharose-6B columns in order to remove the unreacted platinum, and the concentration and the r» of the platinum-DNA complexes were determined.


4.

MACQUET ET AL.

Pt(II) Compounds and DNA In Vitro

81

F i x a t i o n of P t ( I I ) compounds on DNA does not cause c h a i n breaks. A s i n g l e c h a i n break i n s u p e r c o i l e d PM2 DNA converts the polymer from a compact c l o s e d c i r c u l a r conformation t o a r e l a x e d open c i r c u l a r form. This conformational change i s an extremely s e n s i t i v e measure of c h a i n s c i s s i o n and i t has been used to d e t e c t the n i c k i n g of DNA by P t ( I I ) compounds (24, 25, 26), Table I I shows the percentage of n i c k e d PM2 DNA molecules which were observed by e l e c t r o n microscopy a f t e r f i x a t i o n of v a r i o u s amounts of the three p l a t i n u m compounds on the DNA (25), I t can be seen that below rb = 0.1 none of the compounds cause c h a i n breaks i n the DNA. TABLE I I . RATIOS BETWEEN SUPERCOILED AND NICKED PM2 DNA MOLECULES IN THE DIFFERENT PLATINUM-DNA COMPLEXES VISUALIZED BY ELECTRON MICROSCOPY.

Nicked molecules (%)

Compound

r

b

Supercoiled molecules (%)

DNA

0

81(78-84)

19

78(72-84) 73(69-77) 78(77-79) 48(42-54)

22 27 22 52

-2

77(75-79) 80(78-82) 79(76-82) 82(80-84)

23 20 21 18

-2

82(79-85) 80(77-83) 78(74-82) 69(68-70)

18 20 22 31

cis-Pt(NH ) -DNA 3

2

10

-2

IS-

1

trans-?t(NH^) ~DNA 2

10

IS-' Pt(dien)-DNA 10

10

1

Values i n parentheses g i v e the range. 200 molecules of each complex were v i s u a l i z e d at d i f f e r e n t p l a c e s on the g r i d . Cis- and trans-DPP but not [ P t ( d i e n ) C l ] C l form i n t e r s t r a n d c r o s s l i n k s . The best c h a r a c t e r i z e d of the c£s-Pt(NH3)2-DNÀ l e s i o n s i s the i n t e r s t r a n d c r o s s l i n k which has been deduced from the appearance of h i g h molecular weight DNA i n denaturing c o n d i t i o n s (27), enhanced thermal r e n a t u r a t i o n (22) and a d i m i n i s h e d r a t e o f a l k a l i n e e l u t i o n (28). I t i s evident from thermal r e n a t u r a t i o n


82


experiments (Figure 3) that c i s - and trans-DPP, but not [ P t ( d i e n ) C l ] C l form i n t e r s t r a n d c r o s s l i n k s p e r m i t t i n g the reformat i o n of the double h e l i c a l s t r u c t u r e of heat-denatured DNA which i s subsequently cooled (25). Recently Roberts (29) has q u a n t i t a t e d the number of i n t e r s t r a n d c r o s s l i n k s formed by "7s-DDP f o r l x l O " < rb < 5xlO~4. Exposure of e i t h e r p u r i f i e d DNA or DNA i n mammalian c e l l s to c i s - P P P f o r 2 hours produced about 1 c r o s s l i n k per 150 bound p l a t i n u m molecules as determined by the percentage of h i g h molecular weight DNA observed i n a l k a l i n e sucrose g r a d i ents. I n both cases the c r o s s l i n k i n g frequency increased t o about 1/30 when the platinum-DNA complex was s t o r e d i n b u f f e r f o r 24 hours a f t e r treatment. Assuming a c r o s s l i n k i n g frequency of 1/30, the data i n F i g u r e 3 i n d i c a t e s t h a t a s i n g l e c r o s s l i n k per 500-1000 base p a i r s i s necessary f o r complete r e n a t u r a t i o n of the DNA. 5

Cis- and trans-DPP but not [ P t ( d i e n ) C l ] C l shorten DNA. The length of PM2 DNA which has been t r e a t e d by the three compounds has been measured u s i n g e l e c t r o n microscopy (23). This technique appears to be more s e n s i t i v e than m i g r a t i o n d u r i n g g e l e l e c t r o phoresis which d i d not change a p p r e c i a b l y f o r r ^ < 0.05 (30). In c o n t r a s t , f o r r ^ ^ 0.05 e l e c t r o n microscopy r e v e a l e d a l i n e a r r e l a t i o n s h i p between the decrease i n the length of PM2 DNA and the q u a n t i t y of bound cis- or trans-PPP. F i x a t i o n of [ P t ( d i e n ) C l ] C l d i d not change the s i z e of the DNA (Figure 4 ) . Judging from the slopes of the curves i n f i g u r e 4, f i x a t i o n of a s i n g l e cis-DDP shortened the DNA by 17 A w h i l e each trans-PPP decreased the l e n g t h by 10 A. A l t e r a t i o n s of the v i s c o s i t y (25) of DNA as a f u n c t i o n of rb (Figure 5) are remarkably s i m i l a r t o the change i n the l e n g t h of DNA observed u s i n g e l e c t r o n microscopy. The drop i n v i s c o s i t y i s not caused by c h a i n breaks and i s c o n s i s t e n t w i t h the hypothesis that these compounds shorten DNA, o

F i x a t i o n of cis- and trqns-PPP, but not [ P t ( d i e n ) C l ] C l , d i s r u p t s DNA basestacking. Spectroscopic s t u d i e s such as UV hyperchromism and c i r c u l a r d i c h r o i s m r e f l e c t the immediate environment of the base and are s e n s i t i v e probes of conformational changes i n v o l v i n g , at most, s e v e r a l n u c l e i c a c i d bases. Between 0.01 < r < 0.05 cis-DDP increased the p o s i t i v e CD band of DNA at 270 nm by about 40 % (Figure 6) u n l i k e trons-PPP and [ P t ( d i e n ) C l ] C l which had no e f f e c t (31). UV hyperchromism was not observed a f t e r f i x a t i o n of any of the three compounds i n t h i s range of rb (23), A more d i r e c t measurement of the d i s r u p t i o n of base-base i n t e r a c t i o n s by f i x a t i o n of these molecules i s t h e i r a b i l i t y t o prevent the a s s o c i a t i o n of EtdBr w i t h DNA. EtdBr i n t e r c a l a t e s between base p a i r s and the number of i n t e r c a l a t e d molecules can be determined u s i n g f l u o r e s c e n c e spectroscopy (32). C i s - and trans-PPP each prevented the i n t e r c a l a t i o n of one EtdBr molecule per p l a t i n u m bound to DNA w h i l e [ P t ( d i e n ) C l ] C i had no e f f e c t (Figure 7), b


MACQUET ET A L .

Pt(H) Compounds and DNA

ol

, 0.0125

0

, 0.025

In Vitro

, _ 0.050

r

b

Figure 3.

The percentage of renaturation of cis-DDP (Φ),

trans-DDP (M),

and

[Pt(dien)Cl]Cl (A) at low r . 6

Platinum-T7 DNA complexes (25 ng/mL) in NaClO (10 mM) were denatured by raising the temperature to 100°C and then renatured by decreasing the temperature at a rate of 1 °C/min to 25 °C. % renaturation = (A — Α) χ 100/(A — Ao) where A is the absorbance at 260 nm before denaturation, At is the absorbance at 260 nm corre sponding to the maximum of hyperchromicity, and A is the absorbance at 260 nm after renaturation. t

f

f

0

LPNA-Pt

LDNA

50l

ι 0

0.0125

ι 0.025

ι 0.050

r

b

Figure 4.

Shortening of PM2 DNA as a function of r . Key: · , cis-DDP; M, 6

trans-DDP; and A , [Pt(dien)Cl]Cl. Nicked circular PM2 DNA (0.02 mg/mL) was incubated in NaClO (10 mM) with platinum compounds at 20 °C in the dark for 8 d. After dialysis against NaClO (10 mM) and platinum determination by atomic absorption spectrophotometry, the complexes were absorbed on carbon-coated copper grids and observed using a Philips 301 electron microscope. The average length of control DNA (LDNA) and of platinum-DNA complexes (LDNA-PI) were determined from an observation of at least 60 molecules. t

t


Figure 6. Circular-dichroism spectra of salmon sperm DNA alone and complexée with As-DDP {top), trms-DDP (middle), or [Pt(dien)Cl]Cl (bottom) for r = 0.05. 6


MACQUET ET AL.

Pt(II) Compounds and DNA

In Vitro

Figure 7. Number of EtdBr binding sites (η) in salmon sperm DNA complexed with cis-DDP (Φ), trans-DDP (M), or [Pt(dien)Cl]Cl (A) at low r . Scatchard plots of EtdBr binding to different platinum-DNA complexes were used to deter mine n. 6


86


For r b < 0.01, cis-ΌΌ? d e s t a b i l i z e s DNA w h i l e trans-W? and [ P t ( d i e n ) C l ] C l s t a b i l i z e DNA. A l t e r a t i o n o f the m e l t i n g temperat u r e o f DNA by f i x a t i o n of the three platinum compounds a t low r ^ i s shown i n F i g u r e 8. I n a l l cases t h e changes i n T were a l i n e a r f u n c t i o n o f the q u a n t i t y o f bound platinum. For the f i x a t i o n of 5 p l a t i n u m per 1000 bases (30 platinum per T7 DNA molecule), trans-ΌΌΈ and [ P t ( d i e n ) C l ] C l increased the m e l t i n g temperature by 0.7°C and 1.7°C r e s p e c t i v e l y w h i l e e^s-DDP decreased the T by 1.7°C (25). m

m

S i n g l e stranded regions o f DNA are not detected f o r gis-DDP, trans-ΌΌ? o r [ P t ( d i e n ) C l ] C l f o r r < 0.025. I n order t o see i f s i n g l e stranded DNA i s formed by the f i x a t i o n of P t ( I I ) compounds on DNA we have measured the nuclease SI d i g e s t i o n o f salmon sperm DNA which had been t r e a t e d a t low r ^ by the three compounds i n F i g u r e 1. The r e a c t i o n was f o l l o w e d by the r e l e a s e o f a c i d s o l u b l e UV absorbing m a t e r i a l , l i m i t o f d e t e c t i o n 1 % d i g e s t i o n . Nuclease SI from Aspergillus oryzae d i d not d i g e s t DNA which had been t r e a t e d w i t h any of the compounds below r ^ = 0.025. At r ^ = 0,05 cùs-ΏΏ? but not trans-W? or [ P t ( d i e n ) C l ] C l induced s i n g l e stranded regions which were s e n s i t i v e t o t h i s enzyme. At h i g h e r rfc values SI nuclease was able t o p a r t i a l l y d i g e s t DNA which had been t r e a t e d by a l l three compounds. I n a l l cases the extent o f SI nuclease d i g e s t i o n increased n o n l i n e a r l y as a f u n c t i o n o f r ^ . I t appears t h a t the formation o f s i n g l e stranded DNA may be a cooperative phenomenon which r e q u i r e s the p a r t i c i p a t i o n of s e v e r a l f i x e d platinum (33). K

Summary. Physicochemical s t u d i e s of the a l t e r a t i o n o f DNA conformation and s t a b i l i t y by the f i x a t i o n of

Physicochemical and Structural Studies of the In Vitro Interactions

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