DNA as a Target for Anticancer Coordination Compounds - ACS

1 DNA as a Target for Anticancer Coordination Compounds J. J. ROBERTS and M. F. PERA, JR.1

Downloaded by 80.82.77.83 on May 22, 2017 | http://pubs.acs.org Publication Date: January 26, 1983 | doi: 10.1021/bk-1983-0209.ch001

Institute of Cancer Research, Pollards Wood Research Station, Nightingales Lane, Chalfont St. Giles, Bucks, HP8 4SP England The notion that DNA is the likely target for antitumour platinum complexes will be discussed on the basis of their interaction with nucleic acids in vitro and of their interactions with the DNA of both cells in culture and cells in whole animals. Biochemical studies suggest that interactions with cellular DNA result in an inactivation of the DNA template for DNA replication. Support for these views came from the demonstration that cells could remove DNA bound platinum adducts by an excision repair process that facilitated the recovery of cells from toxic damage. Since the first description of the biological properties of platinum complexes there has been a considerable effort to understand the molecular basis of their actions. The clear demonstration of the usefulness of cisplatin in treatment of human tumours has added impetus to this work, because ultimately an understanding of the mechanism of drug action should provide a more rational basis for improvement in therapy. Much evidence is available now to support the view that platinum drugs exert their cytotoxic effects through an interaction with DNA. The main purpose of this review will be to assess critically the evidence supporting this hypothesis. We will first briefly summarise some of the biological properties of platinum compounds other than antitumour activity that are probably a consequence of their ability to react with DNA. Then we will discuss the interaction of platinum compounds with nucleic acids before considering in detail the evidence that indicates DNA as a major target for drug action. 1

Current address: I C R F Laboratories, P O B 123, Lincoln's Inn Fields, London W C 2 A 3 P X England

0097-6156/83/0209-0003$06.75/0 © 1983 American Chemical Society

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

4

METAL CHEMOTHERAPEUTIC AGENTS


B i o l o g i c a l E f f e c t s o f Platinum Antitumour Drugs I n d i c a t i v e o f I n t e r a c t i o n with DNA Probably the f i r s t o b s e r v a t i o n o f an e f f e c t o f a platinum c o o r d i n a t i o n complex i n a b i o l o g i c a l system and one which gave a c l e a r i n d i c a t i o n o f i t s biochemical mode o f a c t i o n was the o b s e r v a t i o n t h a t n e u t r a l platinum c o o r d i n a t i o n complexes i n h i b i t e d b a c t e r i a l c e l l d i v i s i o n and induced the b a c t e r i a t o grow i n t o long f i l a m e n t s . Filamentous growth i n b a c t e r i a may be i n d i c a t i v e o f the a b i l i t y o f an agent t o r e a c t with DNA l e a d i n g t o a s e l e c t i v e i n h i b i t i o n o f DNA s y n t h e s i s , with no accompanying e f f e c t on other b i o s y n t h e t i c pathways such as RNA or p r o t e i n s y n t h e s i s . A v a r i e t y o f agents, such as UV- and X - i r r a d i a t i o n and c y t o t o x i c a l k y l a t i n g agents, can a l s o e l i c i t t h i s response as a r e s u l t o f t h e i r common a b i l i t y t o damage DNA. F u r t h e r important evidence f o r d i r e c t a t t a c k on DNA came from the a b i l i t y o f platinum compounds t o induce the growth o f phage from l y s o g e n i c s t r a i n s o f E . c o l i b a c t e r i a (2). The r e l e a s e o f the phage DNA t o d i r e c t s y n t h e s i s o f new phage i s normally a r a r e event. However, agents which can r e a c t with DNA can cause the phage DNA t o be r e l e a s e d and phage p a r t i c l e s t o be s y n t h e s i z e d with consequent c e l l l y s i s . Reslova (2) was able t o show t h a t there e x i s t s an e x c e l l e n t c o r r e l a t i o n between the antitumour a c t i v i t y of platinum compounds and t h e i r a b i l i t y t o induce l y s o g e n i c Ε.coli t o enter the l y t i c c y c l e . The i n t e r a c t i o n s o f platinum compounds with v i r u s e s have f u r t h e r i n d i c a t e d the r e l a t i v e l y g r e a t e r importance o f r e a c t i o n s with DNA as a g a i n s t those w i t h p r o t e i n i n producing b i o l o g i c a l effects. Kutinova e t a l . (3) demonstrated the i n a c t i v a t i o n o f the i n f e c t i o u s a c t i v i t y o f e x t r a c e l l u l a r papovavirus SV40 by cis-ΡΡΡ. The i n a c t i v a t i o n o f B . s u b t i l i s transforming DNA by platinum compounds, l i k e w i s e i n d i c a t e d the e f f e c t o f these agents on the b i o l o g i c a l f u n c t i o n o f DNA (,30) s t u d i e d the i n t e r a c t i o n o f s e v e r a l platinum compounds w i t h macromolecules a t measured l e v e l s o f c e l l k i l l . To assess the p o s s i b l e importance o f PNA, RNA and p r o t e i n as primary t a r g e t s f o r platinum(II) compounds, these b i n d i n g data (expressed as moles/gm o f macromolecule) were used t o c o n s t r u c t curves o f l o g s u r v i v a l a g a i n s t the amount o f drug bound t o each


8


type o f macromolecule. The r e s u l t i n g graphs were then c h a r a c t e r i s e d i n a manner s i m i l a r t o those r e l a t i n g l o g c e l l s u r v i v a l t o dose o f drug given t o the c e l l s . The shoulder width o f the b i n d i n g curve was given the value Bq and the slope o f the s t r a i g h t l i n e p o r t i o n B . For both c i s and trans-DPP the b i n d i n g c o e f f i c i e n t s were higher f o r RNA than DNA. However, the t r u e s i g n i f i c a n c e o f these b i n d i n g c o e f f i c i e n t s can only be appreciated i f account i s taken o f the molecular weights o f the molecules concerned. I f one assumes no s e l e c t i v i t y i n the b i n d i n g t o any p a r t i c u l a r RNA or p r o t e i n molecule, then i t i s p o s s i b l e t o c a l c u l a t e the number o f platinum molecules bound t o each macromolecule a t a given t o x i c dose. The r e s u l t s o f such a c a l c u l a t i o n , performed a t the c o n c e n t r a t i o n o f cis-DDP which reduced the s u r v i v i n g f r a c t i o n o f Hela c e l l s from f t o 0.37f ( t h i s i s t h e o r e t i c a l l y the c o n c e n t r a t i o n a t which one i n a c t i v a t i n g event occurs, on the average, i n each c e l l ) show the number o f molecules bound t o DNA i s s t r i k i n g l y more than t h a t t o e i t h e r RNA or p r o t e i n , c l e a r l y i n d i c a t i n g t h a t DNA i s the most suscept i b l e c e l l u l a r t a r g e t f o r cis-DDP. The b i n d i n g data f u r t h e r i n d i c a t e t h a t a t t h i s c o n c e n t r a t i o n o f cis-DDP approximately only one molecule o f p r o t e i n out o f 1500 molecules w i l l have r e c e i v e d one p l a t i n a t i o n r e a c t i o n . Unless there i s c o n s i d e r a b l e s p e c i f i c i t y i n the r e a c t i o n o f platinum drugs with a p a r t i c u l a r p r o t e i n enzyme molecule, then t h i s l e v e l o f r e a c t i o n would be too low t o i n a c t i v a t e enzyme a c t i v i t y . Moreover, the l e v e l o f r e a c t i o n with rRNA, tRNA or mRNA would not be expected, again, i n the absence o f any s e l e c t i v i t y o f r e a c t i o n , t o i n a c t i v a t e a l l such molecules and l e a d t o i n t e r f e r e n c e with p r o t e i n s y n t h e s i s . S i m i l a r DNA b i n d i n g and c e l l s u r v i v a l s t u d i e s have been c a r r i e d out i n Chinese hamster c e l l s i n c u l t u r e with a number o f other platinum compounds t h a t have shown encouraging a c t i v i t y against a number o f experimental animal tumours (48). Differences o f up t o t e n f o l d were found i n the molar concentrations o f these agents t h a t were r e q u i r e d t o produce e q u i t o x i c e f f e c t s on c e l l s i n c u l t u r e f o l l o w i n g one hour's i n c u b a t i o n . The l e v e l s o f r e a c t i o n w i t h DNA a t e q u i t o x i c doses ( B v a l u e s ) , on the other hançl, were, f o r most compounds, o f the same order and d i f f e r e d by o n l y a few f o l d (Table I ) .


Q

Q

Reaction with the DNA o f c e l l s i n v i v o . I t i s c l e a r l y e s s e n t i a l , f o r an understanding o f the mechanism o f the tumouri n h i b i t o r y a c t i o n o f platinum compounds, t o e s t a b l i s h that the s e n s i t i v i t y o f tumour c e l l s i n v i v o i s r e l a t e d t o the extent o f r e a c t i o n o f platinum with t h e i r DNA i n a manner s i m i l a r t o t h a t f o r c e l l s t r e a t e d i n v i t r o w i t h these agents. In a p r e l i m i n a r y study, mice b e a r i n g the t r a n s p l a n t e d ADJ/PC6 plasmacytoma were t r e a t e d with cis-DDP and two other a c t i v e platinum congeners, CHIP and cis-diammine(1:1-eyelobutanedicarboxylato)platinum(II) at doses t h a t had an equal i n h i b i t o r y e f f e c t on the tumour (IDgo) (49). Despite the d i f f e r e n c e i n the a c t u a l amounts o f



3

2

3

?

2

2

2

2

2

Cyclobutanedicarboxylic

2

CBDCA

g

Not determined

5

2

13

acid;

120

N.D.

dac, 1,2-diaminocyclohexane

2.4

17.5 65

0.4

14 480

2.5

3.0

23

120

2.5

8.5

N.D.

14.5

48

15

Β D ο ο (μΜ/l h) (nmoles/gm)

Chinese hamster V79 379A c e l l s

N.D.

180

4.2

1.6

54

90 (mg/kg)

I D

50 (mg/kg)

L D

N.D.

2

cis-Pt(II)Cl (C H NH )

4

cis-Pt(II)(SO )H 0(l,2-dac)

cis-Pt(II)(mal)(1,2-dac)

3

cis-Pt(II)(1,1-CBDCA)(NH )

2

cis-Pt(IV)C1 (iso-C H NH ) (OH)

2

cis-Pt(II)Cl (NH )

Compound

ADJ/PC6A Mouse plasmacytoma

Comparison o f the t o x i c i t y o f v a r i o u s platinum complexes towards Chinese hamster V79 379A c e l l s i n c u l t u r e and plasmacytoma ADJ/PC6 tumour c e l l s i n v i v o i n r e l a t i o n t o DNA b i n d i n g (Roberts, 1981)

Table I


w

v©

§

Î

ι

ι

>

α

>

Η

sw


10


the m a t e r i a l s administered t o the mice, the doses d i d not d i f f e r by more than a f a c t o r o f two when expressed i n terms o f t h e i r molar c o n c e n t r a t i o n s . I n t e r e s t i n g l y , the amounts o f the p l a t i num drugs bound t o the tumour DNA a t these e q u i t o x i c concentrat i o n s were a l l remarkably s i m i l a r . Pera e t a l . (50) s t u d i e d the r e a c t i o n o f c i s p l a t i n and hydroxymalonato diammine platinum(II) with DNA o f B16 melanoma and bone marrow i n C57BL mice. I n h i b i t i o n o f tumour growth, and colony formation assays f o r melanoma c e l l s as w e l l as bone marrow stem c e l l s , were used t o q u a n t i t a t e t o x i c i t y and t o i n d i c a t e antitumour s e l e c t i v i t y . Pt[OHmal(NH3)2] produced g r e a t e r s e l e c t i v e i n h i b i t i o n o f tumour growth and more s e l e c t i v e tumour c e l l k i l l i n g compared with c i s p l a t i n . In the case o f c i s p l a t i n , b i n d i n g o f platinum t o DNA a t measured l e v e l s o f s u r v i v a l i n v i v o was s i m i l a r t o values p r e v i o u s l y observed i n c u l t u r e d c e l l s , a f i n d i n g t h a t again strengthens arguments concerning the mechanism o f a c t i o n o f the drugs based on i n v i t r o work. The g r e a t e r s e l e c t i v e t o x i c i t y o f Pt[OHmal(NH3)2) towards the B16 melanoma was a s s o c i a t e d with an i n c r e a s e d b i n d i n g o f platinum t o tumour DNA, r e l a t i v e t o c i s p l a t i n . The enhanced DNA b i n d i n g i n the tumour seen with Pt[OHmal(NH^)2] was not seen i n the marrow (Figure 1 A.B., Table I I ) . Thus the i n c r e a s e d antitumour s p e c i f i c i t y o f the newer congener probably r e s u l t s from pharmacologic f a c t o r s t h a t enhance d e l i v e r y o f a c t i v e drug t o tumour c e l l s . Role o f c r o s s l i n k i n g r e a c t i o n s . The s t r u c t u r a l requirement f o r d i f u n c t i o n a l i t y and the p r i n c i p a l biochemical e f f e c t s o f the platinum compounds, as d i s c u s s e d below, suggest a p a r a l l e l between the platinum drugs and the c l a s s i c a l b i - f u n c t i o n a l a l k y l a t i n g agents such as the n i t r o g e n mustards. The l a t t e r compounds have been thought f o r some time t o produce an i n h i b i t i o n o f DNA s y n t h e s i s by t h e i r a b i l i t y t o introduce c r o s s l i n k s i n t o the DNA o f mammalian c e l l s . I t has, however, been a matter o f contention as t o whether the p r i n c i p a l l e s i o n i s a c r o s s l i n k between strands o f the DNA h e l i x or c r o s s l i n k s between bases on one s t r a n d o f DNA, o r p o s s i b l y , even between DNA and p r o t e i n . C r o s s l i n k i n g was estimated by Roberts and Pascoe (33) from the p r o p o r t i o n o f 'hybrid' DNA formed by l i n k i n g opposing strands o f heavy (BudR l a b e l l e d ) and l i g h t (normal) DNA. The r e l a t i v e t o x i c i t i e s o f the c i s and t r a n s isomers o f the platinum(II) n e u t r a l complexes, can be d e f i n e d by the slopes o f the s u r v i v a l curves (D ) obtained by t r e a t i n g Hela c e l l s i n culture. Comparison o f these two s e t s o f values i n d i c a t e d t h a t the r e l a t i v e a b i l i t i e s o f c i s and trans-DPP compounds t o c r o s s l i n k DNA i n v i v o (but not i n v i t r o ) were r e l a t e d t o t h e i r c y t o t o x i c a c t i o n (29,30). These s t u d i e s t h e r e f o r e suggest t h a t i n t e r s t r a n d c r o s s l i n k i n g with both the platinum(II) compounds, but not n e c e s s a r i l y platinum(IV) compounds, may be important i n i n d u c i n g t h e i r c y t o t o x i c e f f e c t s and t h a t the c i s isomer i s most e f f e c t i v e i n i n d u c i n g the r e a c t i o n . 0


1.

ROBERTS AND PERA

DNA

and Anticancer Compounds

11


35-1

Cisplatin dose (mg/kg)

Β 80-,

70H

0

50

100

150

Pt [ OHMal(NH ) ] dose (mg/kg) 3

2

Figure L Binding of platinum to B16 melanoma DNA (Φ) or bone marrow DNA (A) following treatment of C57B16 mice with cis-diamminedichloroplatinum(II) (ch-DDP) (A) and hydroxymalonatodiammineplatinumfll) (B).



12


Table I I

Amount o f platinum bound t o marrow and tumour DNA of C57BL mice a t doses o f c i s p l a t i n o r Pt[OHMal (NH3) ] producing 37 per cent s u r v i v a l o f bone marrow stem c e l l s (CFU-S) o r B16 lung colony forming c e l l s 2

B16 LCFC

CFU-S

Amount Bound Cisplatin Pt[OHMal(NH ) ] 3

2

Amount Bound

5 mg/Kg

4nmol/g

11 mg/Kg

22nmol/g

40 mg/Kg

5nmol/g

20 mg/Kg

12.5nmol/g



1.

ROBERTS AND PERA

DNA and Anticancer Compounds

13

A r e i n v e s t i g a t i o n o f c r o s s l i n k i n g o f DNA by platinum(II) compounds u s i n g a d i f f e r e n t method from t h a t d e s c r i b e d above, namely a l k a l i n e e l u t i o n (34) has confirmed the g r e a t e r a b i l i t y of cis-DDP as compared with trans-DPP t o c r o s s l i n k c e l l u l a r DNA. These i n v e s t i g a t o r s made the f u r t h e r i n t e r e s t i n g o b s e r v a t i o n t h a t i n c u b a t i o n o f t r e a t e d mouse leukemic L1210 c e l l s i n a drug-free medium r e s u l t e d i n an i n c r e a s e i n the number o f DNA c r o s s l i n k s . C r o s s l i n k i n g e f f e c t s developed, f o l l o w i n g treatment w i t h concent r a t i o n s as low as 1 ym f o r c i s and 5 ym f o r trans-DPP which permitted over 80% s u r v i v a l o f colony-forming a b i l i t y . The maximum c r o s s l i n k i n g e f f e c t by cis-PPP r e q u i r e d about 12 h post treatment i n c u b a t i o n before i t was f u l l y developed by 6 h a f t e r exposure t o the drug. The c r o s s l i n k i n g e f f e c t s o f both agents were reversed upon f u r t h e r i n c u b a t i o n o f the c e l l s , presumably due t o the o p e r a t i o n o f a PNA e x c i s i o n r e p a i r process. A f u r t h e r study employing a l k a l i n e e l u t i o n showed t h a t the c r o s s l i n k i n g e f f e c t produced by c i s and trans-PPP c o u l d be separated i n t o two components, one p r o t e i n a s e - s e n s i t i v e and due t o PNA-protein c r o s s l i n k i n g , another p r o t e i n a s e - r e s i s t a n t and due to PNA i n t e r s t r a n d c r o s s l i n k i n g (51). PNA p r o t e i n c r o s s l i n k s were a t maximum l e v e l s immediately a f t e r drug removal, while PNA-PNA i n t e r s t r a n d c r o s s l i n k s reached maximum l e v e l s 6-12 hours a f t e r drug removal. T o x i c i t y o f the two agents i n L1210 leukemia c e l l s , and V79 Chinese hamster c e l l s , c o r r e l a t e d w e l l with PNA i n t e r s t r a n d c r o s s l i n k i n g , but not with PNA-protein crosslinking Because the b i o p h y s i c a l b a s i s o f the e l u t i o n technique i s p o o r l y understood, q u a n t i t a t i o n o f i n t e r s t r a n d c r o s s l i n k i n g with t h i s technique i s based upon l a r g e l y unproven assumptions. Therefore s t u d i e s were undertaken t o compare e l u t i o n r e s u l t s with the a l k a l i n e caesium c h l o r i d e technique d e s c r i b e d above, a l k a l i n e sucrose sedimentation, and e s t i m a t i o n o f renaturable PNA i n c e l l l y s a t e s . D i r e c t q u a n t i t a t i o n o f c r o s s l i n k frequency f o l l o w i n g c i s p l a t i n treatment was thus obtained over a wide dose range u s i n g methods based upon known b i o p h y s i c a l p r o p e r t i e s o f PNA. The r e s u l t s showed c l e a r l y t h a t a l k a l i n e e l u t i o n f o l l o w i n g p r o t e i n a s e d i g e s t i o n gave an accurate measure o f i n t e r s t r a n d c r o s s l i n k i n g , and showed a l s o t h a t c r o s s l i n k s were o n l y a small f r a c t i o n o f the t o t a l drug-PNA r e a c t i o n products I t has been shown t h a t c e l l s can be p r o t e c t e d from the t o x i c e f f e c t s o f cis-DDP by p r e v e n t i n g the formation o f PNA c r o s s l i n k s by i n c u b a t i n g c e l l s i n the presence o f t h i o u r e a immediately a f t e r treatment (54), a f i n d i n g which f u r t h e r supports a c y t o t o x i c r o l e f o r PNA i n t e r s t r a n d c r o s s l i n k s . F u r t h e r i n v e s t i g a t i o n i n mouse leukemia c e l l s and human f i b r o b l a s t s o f v a r y i n g s e n s i t i v i t y t o c i s p l a t i n , have shown t h a t c e l l u l a r s e n s i t i v i t y o f t e n c o r r e l a t e s with i n t e r s t r a n d c r o s s l i n k formation (55, 56, 57). However, studies o f c e r t a i n mouse leukemia L1210 lines resistant to c i s platin as w e l l as s t u d i e s o f Walker carcinoma c e l l s

(9,51).

(52,53).

(55,58),


(59)

14


have i n d i c a t e d t h a t there i s not always a simple c o r r e l a t i o n between c r o s s l i n k formation and c e l l k i l l . I t i s e n t i r e l y p o s s i b l e t h a t i n t r a s t r a n d c r o s s l i n k i n g might provide an even b e t t e r c o r r e l a t i o n , but i t i s not p o s s i b l e to measure t h i s l e s i o n i n mammalian c e l l s at the present time.


Biochemical E f f e c t s of Drug-DNA

Interaction

I n h i b i t i o n o f DNA s y n t h e s i s . The s i g n i f i c a n c e of the i n t e r a c t i o n of platinum compounds with c e l l u l a r DNA i s apparent from s t u d i e s of drug e f f e c t s on macromolecular s y n t h e s i s . Cis-DDP s e l e c t i v e l y and p e r s i s t e n t l y i n h i b i t s the r a t e of DNA s y n t h e s i s as compared with e f f e c t s on RNA and p r o t e i n s y n t h e s i s i n c e l l s i n c u l t u r e (60-63) and c e l l s i n v i v o (64,65). The l i k e l y b a s i s f o r t h i s s e l e c t i v e biochemical e f f e c t on DNA s y n t h e s i s came from the o b s e r v a t i o n t h a t the i n h i b i t i o n of DNA s y n t h e s i s was p e r s i s t e n t and p r o g r e s s i v e with time a f t e r removal of the drug. I t i s now c l e a r , p a r t i c u l a r l y by com p a r i s o n with analogous e f f e c t s produced by d i r e c t r e a c t i n g agents such as mustard gas, t h a t both e f f e c t s are c o n s i s t e n t with the view t h a t the primary chemical l e s i o n i s i n the DNA of the c e l l , which i s then i n h i b i t e d as a template f o r DNA r e p l i c a t i o n . Thus m o d i f i c a t i o n s to the DNA template w i l l block DNA r e p l i c a t i o n but not a f f e c t t r a n s c r i p t i o n or t r a n s l a t i o n . Under c o n d i t i o n s of low c e l l k i l l i n g , the s e l e c t i v e i n h i b i t i o n by platinum com pounds o f DNA s y n t h e s i s but not of RNA or p r o t e i n s y n t h e s i s , leads to the formation of g i a n t c e l l s , a feature observed i n c e l l s t r e a t e d with a v a r i e t y o f agents a l s o known to block DNA replication selectively. Studies o f synchronized V79 Chinese hamster c e l l s t r e a t e d i n Gl w i t h c i s p l a t i n showed t h a t depression of DNA s y n t h e s i s i n these c e l l s was the r e s u l t of a decrease i n DNA s y n t h e t i c r a t e , r a t h e r than a decreased r a t e o f entry of c e l l s i n t o S (62). As a r e s u l t , S phase was prolonged i n these c e l l s (Figure 2). F o l l o w i n g Gl treatment with c i s p l a t i n , synchronized Hela c e l l s a l s o showed a decrease i n the amount of thymidine i n c o r p o r a t i o n i n t o DNA d u r i n g S phase, but the e f f e c t was not immediately manifested i n these c e l l s (63) (Figure 3). Thus c e l l s d i f f e r i n the way i n which t h e i r r e p l i c a t i o n machinery responds to c i s p l a t i n - i n d u c e d damage. Such d i f f e r e n c e s might account f o r v a r i a t i o n s i n c e l l u l a r s e n s i t i v i t y (see below) and f u r t h e r s t u d i e s i n t h i s area are warranted. Johnson et a l . (66) used an i n v i t r o T7 DNA r e p l i c a t i o n system which copies exogenous T7 DNA by a mechanism t h a t c l o s e l y mimics i n v i v o DNA r e p l i c a t i o n to demonstrate the r e l a t i v e i n h i b i t o r y e f f e c t of e i t h e r p y r i m i d i n e dimers or bound 195mpt:l a b e l l e d c i s or trans-DPP. I t c o u l d be shown t h a t cis-DDP and pyrimidine dimers i n h i b i t e d DNA r e p l i c a t i o n to the same extent per l e s i o n (63% i n h i b i t i o n per 3 χ 10"*^ l e s i o n s / n u c l e o t i d e phos phate) and the trans-DPP was 5 - f o l d l e s s i n h i b i t o r y .



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Τ

DNA

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Anticancer Compounds

15

Time after harvest (hours)

Figure 2. Effects of cis-DDP treatment in Gl phase on DNA synthesis in synchro nized Chinese hamster V79 cells. Key to cis-DDP concentration: · , 0; A> 1.0 μΜ; 5.0 /tM; and 10.0 μΜ. Τ indicates time of treatment.

Time after harvest (hours) Figure 3. Effects of cis-DDP treatment in Gl phase on DNA synthesis in synchro nized Hela cells. Key to cis-DDP concentration: · , 0; Δ , 0.1 μΜ; • , 0.25 μΜ; A , 1.0 μΜ; and Ο , 2.0 Μ. μ


16



The a l t e r n a t i v e p o s s i b i l i t y t h a t DNA synthesis i s i n h i b i t e d because o f the i n a c t i v a t i o n o f enzymes i n v o l v e d i n DNA r e p l i c a t i o n seems c o n t r a - i n d i c a t e d , not only by the f a i l u r e o f cis-DDP to block p r o t e i n s y n t h e s i s , but a l s o by i t s f a i l u r e t o i n a c t i v a t e DNA polymerase i n v i t r o except with very high concentrations (67). R e l a t i o n between c a p a c i t y t o r e p l i c a t e on a damaged template and c y t o t o x i c i t y . Further evidence r e l a t i n g drug induced a l t e r a t i o n s i n DNA s y n t h e s i s t o c y t o t o x i c i t y i n b a c t e r i a and mammalian c e l l s comes from s t u d i e s o f how c e l l s cope with DNA template damage. Studies o f e x c i s i o n r e p a i r o f platinum induced damage, discussed below, i n d i c a t e t h a t the m a j o r i t y o f DNAplatinum products, i n v o l v i n g one strand o f a double h e l i x , are chemically s t a b l e and are only slowly removed from DNA by an enzymatic process. Thus i t could be supposed t h a t p e r s i s t e n t l e s i o n s i n DNA are circumvented during DNA r e p l i c a t i o n by a process which i s analogous t o t h a t which f a c i l i t a t e s the s u r v i v a l of e x c i s i o n d e f e c t i v e b a c t e r i a a f t e r U V - i r r a d i a t i o n . I t now seems c e r t a i n t h a t mammalian c e l l s a l s o possess v a r y i n g c a p a c i t i e s t o r e p l i c a t e t h e i r DNA on a template c o n t a i n i n g unexcised damage. I t i s a l s o apparent, however, t h a t the mechanism o f any such r e p a i r process d i f f e r s from t h a t which i s thought t o occur i n b a c t e r i a . However, i r r e s p e c t i v e o f the mechanism i n v o l v e d i n s y n t h e s i s i n g past r a d i a t i o n o r c h e m i c a l l y induced l e s i o n s i n DNA, i t has been found t h a t i n some c e l l s the process i s amenable t o i n h i b i t i o n by the trimethylxanthine, caffeine. Thus i t has been shown t h a t the r a t e o f elongation o f newly synthesised DNA i n U V - i r r a d i a t e d o r c h e m i c a l l y - t r e a t e d c e l l s , was d r a m a t i c a l l y impaired i n the presence o f c a f f e i n e . As a consequence o f t h i s i n h i b i t i o n , many c e l l l i n e s , competent i n t h i s r e p l i c a t i v e by-pass, are rendered extremely s e n s i t i v e t o the l e t h a l e f f e c t s o f these agents by post treatment incubation i n the presence o f non-toxic concentrations o f c a f f e i n e (68). There i s now ample evidence t o i n d i c a t e t h a t UV- o r X - i r r a d i a t i o n o r chemically-induced c e l l death i s a f u n c t i o n o f the amount o f chromosome damage which can be observed a t the f i r s t or second m i t o s i s a f t e r treatment. Post treatment incubation i n the presence o f c a f f e i n e enhances d r a m a t i c a l l y the chromosome damaging e f f e c t s o f U V - i r r a d i a t i o n and chemicals i n both p l a n t and animal c e l l s . The v a r i o u s c e l l u l a r e f f e c t s o f cis-DDP and t h e i r m o d i f i c a t i o n s by c a f f e i n e , suggest t h a t l e s i o n s are introduced onto DNA by platinum compounds which are a l s o circumvented by a c a f f e i n e s e n s i t i v e process (69). Post treatment i n c u b a t i o n o f c e l l s i n medium c o n t a i n i n g 0.75 mM c a f f e i n e d r a m a t i c a l l y p o t e n t i a t e d the t o x i c i t y o f c i s p l a t i n and increased the number o f c e l l s c o n t a i n i n g chromosome damage. C a f f e i n e not only increased the number o f cis-DDP t r e a t e d c e l l s c o n t a i n i n g chromosomal aberrations but i t a l s o enhanced the s e v e r i t y o f the damage observed. The delayed appearance o f chromosome abnormali t i e s a f t e r cis-DDP treatment a l s o suggests t h a t DNA r e p l i c a t i o n



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i s necessary f o r t h e i r formation, and i n t h i s r e s p e c t cis-DDP resembles U V - i r r a d i a t i o n and a l k y l a t i n g agents r a t h e r than X - i r r a d i a t i o n (69). The proposal has t h e r e f o r e been made t h a t inadequate r e p l i c a t i o n o f DNA on a damaged template i s r e s p o n s i b l e f o r both c e l l death and chromosome damage and t h a t post treatment i n c u b a t i o n o f c e l l s i n medium c o n t a i n i n g c a f f e i n e , enhances these two e f f e c t s o f DNA damage by i n h i b i t i n g a process which would permit r e p l i c a t i o n to proceed past l e s i o n s . Support f o r t h i s n o t i o n has come from s t u d i e s on both the r a t e o f DNA s y n t h e s i s and the s i z e o f DNA synthesised i n both asynchronous and synchronised populations o f cis-DDP t r e a t e d c e l l s i n the presence and absence o f c a f f e i n e (61,62). I t was found t h a t post treatment i n c u b a t i o n i n medium c o n t a i n i n g a non-toxic c o n c e n t r a t i o n o f c a f f e i n e , reversed the cis-DDP induced i n h i b i t i o n o f DNA s y n t h e s i s i n asynchronous popul a t i o n s o f c e l l s (61). The s i z e of newly synthesised DNA i n cis-DDP t r e a t e d c e l l s may be c o n t r a s t e d with the s i z e o f such DNA i n c e l l s t r e a t e d s i m i l a r l y with cis-DDP and l a b e l l e d with. (^H)TdR i n the presence o f non-toxic concentrations o f c a f f e i n e . Under these c o n d i t i o n s the s i z e o f nascent DNA was markedly reduced as compared with t h a t i n untreated c o n t r o l or cis-DDP only t r e a t e d c e l l s . The s i z e o f the DNA synthesised d u r i n g 4 hours i n the presence o f c a f f e i n e i n cis-DDP t r e a t e d c e l l s was dependent on the i n i t i a l dose o f DPP. I t thus appears t h a t c a f f e i n e i n t e r f e r e s with the mechanism by which the c e l l r e p l i c ates i t s DNA past l e s i o n s on the DNA template. Some support f o r t h i s n o t i o n was obtained from a comparison o f the d i s t a n c e between platinum-induced l e s i o n s on the template strand o f DNA and the s i z e o f the newly s y n t h e s i s e d DNA i n c e l l s t r e a t e d with v a r i o u s doses o f cis-PPP and post incubated i n the presence o f caffeine. The d i s t a n c e between platinum atoms on one strand o f DNA was c a l c u l a t e d from atomic absorption measurements o f the platinum bound to DNA i s o l a t e d from cis-DDP t r e a t e d c e l l s and t h i s was found to correspond c l o s e l y to the s i z e o f the newlysynthesised DNA. I t was concluded, t h e r e f o r e , t h a t i n Chinese hamster c e l l s a l l unexcised p l a t i n a t i o n r e a c t i o n s are normally circumvented d u r i n g DNA r e p l i c a t i o n by a c a f f e i n e - s e n s i t i v e process. R e l a t i o n s h i p Between E x c i s i o n Repair o f Platinum-induced Damage and C y t o t o x i c i t y

PNA

F r a v a l and Roberts (70) demonstrated removal o f platinum adducts from DNA o f e x p o n e n t i a l l y growing Chinese hamster V79 cells. The h a l f - l i f e o f t o t a l drug-DNA r e a c t i o n products was approximately 28 hours. As such products are s t a b l e chemically under p h y s i o l o g i c a l c o n d i t i o n s , removal o f the DNA adducts could be a t t r i b u t e d to r e p a i r . Recent i n v e s t i g a t i o n s using a l k a l i n e e l u t i o n (51_) or a comb i n a t i o n o f a l k a l i n e e l u t i o n , a l k a l i n e sucrose sedimentation and



18


DNA r e n a t u r a t i o n , (53) have c l e a r l y demonstrated r e p a i r o f DNAp r o t e i n and DNA-DNA i n t e r s t r a n d c r o s s l i n k s i n c u l t u r e d mammalian c e l l s f o l l o w i n g c i s p l a t i n treatment. Interstrand c r o s s l i n k i n g i n Chinese hamster V79 c e l l s was demonstrated by a decrease i n the r a t e o f f i l t e r e l u t i o n o f DNA from X - i r r a d i a t e d , t r e a t e d c e l l s , a s h i f t i n a l k a l i n e sucrose g r a d i e n t s o f DNA molecules towards the high molecular weight end o f the gradient, o r an increase i n the r a p i d l y r e n a t u r i n g f r a c t i o n o f DNA i n c e l l l y s a tes. A l l o f these drug-induced phenomena reached a maximum from 6-12 hours a f t e r drug treatment, then d e c l i n e d . The h a l f l i f e o f DNA i n t e r s t r a n d c r o s s l i n k s u s u a l l y appeared t o be between 12-24 hours. Some DNA degradation occurred, but i t was i n s u f f i c i e n t t o account f o r c r o s s l i n k r e v e r s a l . Because c r o s s l i n k s induced by platinum are s t a b l e i n i s o l a t e d DNA under p h y s i o l o g i c a l c o n d i t i o n s , t h i s r e v e r s a l may a l s o be a t t r i b u t e d t o r e p a i r , though the mechanism remains unknown. Although c o r r e l a t i o n s have been e s t a b l i s h e d i n some cases between the extent o f drug-DNA i n t e r a c t i o n and c y t o t o x i c i t y , the r e l a t i o n s h i p between e x c i s i o n r e p a i r o f DNA damage and t o x i c i t y i s not always c l e a r . The rare s k i n c o n d i t i o n , Xeroderma pigmentosum (XP), i s c h a r a c t e r i s e d by extreme s e n s i t i v i t y t o s u n l i g h t and a p r e d i s p o s i t i o n t o s k i n cancer. C e l l s taken from persons s u f f e r i n g from t h i s c o n d i t i o n are more s e n s i t i v e t o UV-irradiation than normal c e l l s and are d e f i c i e n t i n e x c i s i o n r e p a i r o f UV-induced damage. These same c e l l s are a l s o s e n s i t i v e t o other DNA damaging agents such as hydrocarbon epoxides, 4-nitroquinoline-l-oxide and 7-bromomethylbenz(a)-anthracene and s e n s i t i v i t y i s again a s s o c i a ted with decreased l e v e l s o f v a r i o u s m a n i f e s t a t i o n s o f DNA excision repair. (For review see (68). I t has now been found (71) t h a t these r e p a i r d e f i c i e n t XP c e l l s are a l s o more s e n s i t i v e than normal foetus lung c e l l s t o cis-DDP, when the l e t h a l e f f e c t s o f the drug are expressed as a f u n c t i o n o f r e a c t i o n with DNA r a t h e r than as a f u n c t i o n o f dose o f reagent. I t could t h e r e f o r e be reasoned t h a t t h i s increased s e n s i t i v i t y o f XP c e l l s i s s i m i l a r l y due t o t h e i r decreased a b i l i t y t o excise cis-DDP induced DNA damage. C e l l s from p a t i e n t s with the genetic disease Fanconi's anaemia, show unusual s e n s i t i v i t y t o the c y t o t o x i c and c l a s t o g e n i c e f f e c t s o f d i f u n c t i o n a l a l k y l a t i n g agents. These c e l l s are a l s o unusually s e n s i t i v e t o c i s p l a t i n (72). Such s e n s i t i v i t y i s not the r e s u l t o f decreased binding o f platinum t o DNA but i t remains t o be determined i f r e p a i r o f v a r i o u s l e s i o n s i n these c e l l s i s abnormal. I f DNA synthesis on a damaged template i s responsible f o r t o x i c i t y , c e l l s t h a t are allowed t o r e p a i r DNA p r i o r t o S-phase and c e l l d i v i s i o n should show l e s s t o x i c i t y than c e l l s e n t e r i n g the p r o l i f e r a t i v e c y c l e immediately f o l l o w i n g treatment. In an i n i t i a l study, F r a v e l and Roberts (70) t r e a t e d s t a t i o n a r y Chinese hamster V79 c e l l s with c i s p l a t i n and measured t o x i c i t y and p l a t -



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inum-DNA i n t e r a c t i o n a f t e r v a r i o u s h o l d i n g p e r i o d s i n the nondividing state. The c e l l s slowly excised platinum, and p l a t i n g e f f i c i e n c y increased. There was a s i m i l a r r e l a t i o n s h i p between the amount o f platinum bound t o DNA and c e l l s u r v i v a l whether one compared the two immediately f o l l o w i n g treatment with s e v e r a l drug doses, o r a f t e r v a r y i n g p e r i o d s o f recovery. However, the c e l l s used i n these i n i t i a l s t u d i e s , Chinese hamster V79 c e l l s , do not t o l e r a t e the n o n d i v i d i n g s t a t e w e l l , and i t was l a t e r found t h a t they do not recover from c i s p l a t i n t o x i c i t y under some c o n d i t i o n s . Therefore, the experiment was repeated using human f i b r o b l a s t s (73) under c o n d i t i o n s where minimal DNA synthesis occurred. These c e l l s were healthy i n the nondividing condition. The f i b r o b l a s t s recovered from c i s p l a t i n t o x i c i t y i f h e l d i n the n o n d i v i d i n g s t a t e , and they excised platinum l e s i o n s from DNA by a f i r s t order process with a h a l f l i f e o f 2.5 days (Figure 4 ) . DNA-DNA i n t e r s t r a n d c r o s s l i n k s , measured by a l k a l i n e e l u t i o n and by e s t i m a t i o n o f renaturable DNA i n c e l l l y s a t e s , were found t o be r e p a i r e d with a h a l f - l i f e o f about 36 hours (Figure 5). DNA p r o t e i n c r o s s l i n k s were removed at a s i m i l a r r a t e and there was no evidence f o r any accompanying degradation. Thus the r e v e r s a l o f c r o s s l i n k i n g was a t t r i b u t e d to r e p a i r rather than i n t r o d u c t i o n o f DNA strand breaks. The r e l a t i o n s h i p between c e l l s u r v i v a l and the amounts o f platinum remaining bound t o DNA a t the time c e l l s were p l a t e d out f o r e s t i m a t i o n o f c e l l s u r v i v a l , was s i m i l a r t o that observed i n c e l l s t r e a t e d with s e v e r a l drug doses and p l a t e d immediately. The r e s u l t s s t r o n g l y supported the hypothesis that damage present on the DNA template a t the time o f entry i n t o the p r o l i f e r a t i v e c y c l e s was r e s p o n s i b l e f o r c e l l u l a r t o x i c i t y . F u r t h e r , the r e s u l t s showed t h a t the r e p a i r process was a c t u a l l y e f f e c t i v e i n achieving b i o l o g i c a l recovery. When, however, attempts have been made t o r e l a t e inherent drug s e n s i t i v i t y t o r e p a i r c a p a c i t y , c l e a r c u t r e s u l t s have not always emerged. Walker 256 carcinoma c e l l s d i s p l a y unusual s e n s i t i v i t y t o d i f u n c t i o n a l a l k y l a t i n g agents and t o c i s p l a t i n . There i s a s u b l i n e o f t h i s tumour t h a t shows a 30-fold increase in resistance to c i s p l a t i n . Nevertheless, the two s u b l i n e s bind the same amount o f platinum on DNA f o l l o w i n g treatment with a given dosage, they excise platinum l e s i o n s from t h e i r DNA a t a s i m i l a r r a t e , and they remove DNA-protein c r o s s l i n k s and DNA i n t e r s t r a n d c r o s s l i n k s from DNA a t a s i m i l a r r a t e (74). Followi n g sulphur mustard treatment, nascent DNA synthesis on a damaged template appears t o be s i m i l a r i n both l i n e s (75). I t i s of course p o s s i b l e t h a t these l i n e s might vary i n t h e i r c a p a c i t y t o remove a s p e c i f i c l e s i o n t h a t was not measured - an i n t r a s t r a n d c r o s s l i n k , f o r example - b u t i f t h i s i s not the case, then some t a r g e t other than simply DNA e x i s t s i n c e r t a i n c e l l s that confers unusual s e n s i t i v i t y t o b i f u n c t i o n a l agents. T h i s could conceivably be a t the l e v e l o f chromatin o r a DNA-nuclear matrix complex.


20


loo-.

Δ


• Ο

Δ

S

Ο 6S

•

i2

H

o.i2

4

6

Time post treatment (days) Figure 4a. Recovery of nondividing human fibroblasts from cis-DDP toxicity. Stationary-phase humanfibroblastswere treated with cis-DDP (A O , 32 μΜ; and A • • · , 40 μΜ) and were either plated immediately or held in the nondividing state for various time periods and recovered from drug toxicity.


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1.

Figure 4b. Excision of Pt from DNA of nondividing humanfibroblastsfollowing cis-DDP treatment. The plot illustratesfirst-orderloss of the Pt reaction products.




Figure 5. Repair of cis-DDP-induced DNA-protein crosslinks and DNA interstrand crosslinks in humanfibroblastsduring holding in the nondividing state. Stationary cultures were treated with 40 μτηοΐ cis-DDP, and after various periods of holding in the nondividing state, crosslinking was measured by alkaline elution (W, DNA-protein crosslinks; and O, DNA interstrand crosslinks) or by estimation of renaturable DNA in cell lysates (Φ, DNA interstrand crosslinks).


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Literature Cited 1. 2. 3. 4. 5.


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