68P
STUDIES ON THE MODE OF A C T ~ O N OF THE A N T I T U M O U R D R U G N I T R O G E N M ~ S T A R D(MUSTINE) G . E . Spurgin and J . A . Hickman, Cancer Chemotherapy Group, Department of Pharmacy, U n i v e r s i t y o f Aston, Birmingham 84 7ET. I t i s g e n e r a l l y c o n s i d e r e d t h a t the mechanism o f a c t i o n of a n t i t u m o u r d i f u n t i o n a l a l k y l a t i n g a g e n t s , such a s n i t r o g e n mustard ( I ) , i n v o l v e s c o v a l e n t r e a c t i o n w i t h and c r o s s - l i n k a g e of c e l l u l a r DNA, an e v e n t which p r e v e n t s c e l l r e p l i c a t i o n (Connors, 1 9 7 1 ) . T h i s h y p o t h e s i s may n o t be a b l e t o f u l l y e x p l a i n t h e antiturnour e f f e c t s of t h i s c l a s s o f a g e n t and o t h e r o r a d d i t i o n a l t a r g e t s have been Suggested (Wheeler, 1 9 6 7 ) . W e have i n v e s t i g a t e d t h e mechanism o f i n h i b i t i o n o f tumour c e l l r e p l i c a t i o n caused by n i t r o g e n mustard by a s t u d y o f i t s e f f e c t on c e l l memb r a n e enzymeswhich c o n t r o l t h e t r a n s p o r t of i o n s . Changes i n i o n f l u x , p a r t i c u l a r l y t h o s e brought a b o u t by t h e a c t i o n of Na+K+ ATPase and Mg2+ ATPase, a r e c o n s i d e r e d t o r e g u l a t e c e l l d i v i s i o n (Sanui and Rubin, 1978; Dornand e t a 1 1978) and w e c o n s i d e r e d t h a t t h e c y t o t o x i c a c t i o n of n i t r o g e n mustard may be e x p l a i n e d i f i t i n h i b i t e d t h e s e enzymes. ATPases are s e n s i t i v e t o a l k y l a t i n g a g e n t s (Skou 1 9 6 3 ) , are s u b - u n i t enzymes and s o may be c r o s s - l i n k e d (Sweadner, 1 9 7 7 ) , and have e l e v a t e d a c t i v i t y i n t r a n s f o r m e d c e l l s when compared t o normal c e l l s (Kasorov and Friedman, 1 9 7 4 ) . ,CH CH C 1 ,CH2CH2Cl 2 2 CH - N CH3- N 3 \ CH2CH2Cl CH
,
~
(1)
(11)
S t u d i e s were made on t h e ADJ/PC6 plasma tumour, a t u m o u r s e n s i t i v e t o a l k y l a t i n g The e f f e c t s o f n i t r o g e n mustard on t h e ATPase a c t i v i t y of b o t h whole agents. c e l l s and a membrane f r a c t i o n w a s determined and t h e s e e f f e c t s c o r r e l a t e d with a n e s t i m a t e of c y t o t o x i c i t y u s i n g a b i o a s s a y system. N a + K + ATPase ( E .C. 3.6.1.3) , Mg2+ ATPase, e x t e r n a l C a 2 + ATPase and E-nitrophenolphosphatase ( E . C . 3 . 6 . 3 .I) a c t i v i t i e s were c h a r a c t e r i s e d i n t h e crude c e l l membrane p r e p a r a t i o n . Nitrogen mustard i n h i b i t e d 90% of N a + ~ATPase a t 9 x 1O-l5M and Mg2+ ATPase a t 7 x l O - l h i n a time dependant manner b u t had l e s s e f f e c t on e i t h e r t h e e x t e r n a l Ca2+ ATPase o r p-nitrophenolphosphatase a c t i v i t i e s ( I D g O ' s > . A monofunctional analogue (11) o f n i t r o g e n mustard which h a s no antitumour e f f e c t and which o f 8 x 1 0 T 6 M on Na+K+ ATPase and o f 5 x 1 0 - 6 M on c a n n o t c r o s s - l i n k , had I D 90 Mg2+ ATPase. The e f f e c t o f n i t r o g e n mustard on whole PC6 c e l l s w a s determined by e s t i m a t i n g t h e a b i l i t y of c e l l s g i v e n a h y p o t o n i c shock t o t h e n reduce t h e i r c e l l volume (as measured by a C o u l t e r Counter C h a n n e l y s e r ) , a p r o c e s s dependent upon t h e a c t i v i t y o f Na+K+ ATPase (Shank and Smith, 1 9 7 6 ) . N i t r o g e n mustard (lo-',) c o m p l e t e l y i n h i b i t e d t h e r e c o v e r y from hypotonic shock and t h i s c o n c e n t r a t i o n c o r r e s p o n d s t o t h a t which, under i d e n t i c a l i n c u b a t i o n c o n d i t i o n s , caused >90% The monofunctional analogue (11) w a s i n a c t i v e a t lo-% cell k i l l i n t h e bioassay. I t i s concluded t h a t t h e i n h i b i t i o n o f c e l l membrane ATPases by n i t r o g e n mustard may be an i m p o r t a n t f a c e t o f i t s mechanism o f antitumour a c t i o n .
Connors, T.A. ( 1 9 7 1 ) i n Fundamentals o f Biochemical Pharmacology ( e d . Z.M. p461 Pergamon P r e s s , Oxford. Dornand, J. e t a1 (1978) Biochim. Biophys. Acta, 509: 194-200 Kasorov, L . B . and Friedman, H . (1974) Cancer Res., 34: 1862-1865 S a n u i , H . and Rubin, A.H. ( 1 9 7 8 ) , J . C e l l P h y s i o l . 96: 265-278 ( 1 9 7 6 ) , J . C e l l P h y s i o l . 87: 377-387 Shank, B.B. and Smith, N.E. Skou, J.C.(1963) , Biochem. Biophys. R e s . Commun. 10: 79-84. Sweadner, K . J . (1977) Biochem. Biophys. Res. Corn-un. 78: 962-969 Wheeler, G . P . (1967) Fed. Proc. 26: 885-892.
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STUDIES ON THE MODE OF A C T ~ O N OF THE A N T I T U M O U R D R U G N I T R O G E N M ~ S T A R D(MUSTINE) G . E . Spurgin and J . A . Hickman, Cancer Chemotherapy Group, Department of Pharmacy, U n i v e r s i t y o f Aston, Birmingham 84 7ET. I t i s g e n e r a l l y c o n s i d e r e d t h a t the mechanism o f a c t i o n of a n t i t u m o u r d i f u n t i o n a l a l k y l a t i n g a g e n t s , such a s n i t r o g e n mustard ( I ) , i n v o l v e s c o v a l e n t r e a c t i o n w i t h and c r o s s - l i n k a g e of c e l l u l a r DNA, an e v e n t which p r e v e n t s c e l l r e p l i c a t i o n (Connors, 1 9 7 1 ) . T h i s h y p o t h e s i s may n o t be a b l e t o f u l l y e x p l a i n t h e antiturnour e f f e c t s of t h i s c l a s s o f a g e n t and o t h e r o r a d d i t i o n a l t a r g e t s have been Suggested (Wheeler, 1 9 6 7 ) . W e have i n v e s t i g a t e d t h e mechanism o f i n h i b i t i o n o f tumour c e l l r e p l i c a t i o n caused by n i t r o g e n mustard by a s t u d y o f i t s e f f e c t on c e l l memb r a n e enzymeswhich c o n t r o l t h e t r a n s p o r t of i o n s . Changes i n i o n f l u x , p a r t i c u l a r l y t h o s e brought a b o u t by t h e a c t i o n of Na+K+ ATPase and Mg2+ ATPase, a r e c o n s i d e r e d t o r e g u l a t e c e l l d i v i s i o n (Sanui and Rubin, 1978; Dornand e t a 1 1978) and w e c o n s i d e r e d t h a t t h e c y t o t o x i c a c t i o n of n i t r o g e n mustard may be e x p l a i n e d i f i t i n h i b i t e d t h e s e enzymes. ATPases are s e n s i t i v e t o a l k y l a t i n g a g e n t s (Skou 1 9 6 3 ) , are s u b - u n i t enzymes and s o may be c r o s s - l i n k e d (Sweadner, 1 9 7 7 ) , and have e l e v a t e d a c t i v i t y i n t r a n s f o r m e d c e l l s when compared t o normal c e l l s (Kasorov and Friedman, 1 9 7 4 ) . ,CH CH C 1 ,CH2CH2Cl 2 2 CH - N CH3- N 3 \ CH2CH2Cl CH
,
~
(1)
(11)
S t u d i e s were made on t h e ADJ/PC6 plasma tumour, a t u m o u r s e n s i t i v e t o a l k y l a t i n g The e f f e c t s o f n i t r o g e n mustard on t h e ATPase a c t i v i t y of b o t h whole agents. c e l l s and a membrane f r a c t i o n w a s determined and t h e s e e f f e c t s c o r r e l a t e d with a n e s t i m a t e of c y t o t o x i c i t y u s i n g a b i o a s s a y system. N a + K + ATPase ( E .C. 3.6.1.3) , Mg2+ ATPase, e x t e r n a l C a 2 + ATPase and E-nitrophenolphosphatase ( E . C . 3 . 6 . 3 .I) a c t i v i t i e s were c h a r a c t e r i s e d i n t h e crude c e l l membrane p r e p a r a t i o n . Nitrogen mustard i n h i b i t e d 90% of N a + ~ATPase a t 9 x 1O-l5M and Mg2+ ATPase a t 7 x l O - l h i n a time dependant manner b u t had l e s s e f f e c t on e i t h e r t h e e x t e r n a l Ca2+ ATPase o r p-nitrophenolphosphatase a c t i v i t i e s ( I D g O ' s > . A monofunctional analogue (11) o f n i t r o g e n mustard which h a s no antitumour e f f e c t and which o f 8 x 1 0 T 6 M on Na+K+ ATPase and o f 5 x 1 0 - 6 M on c a n n o t c r o s s - l i n k , had I D 90 Mg2+ ATPase. The e f f e c t o f n i t r o g e n mustard on whole PC6 c e l l s w a s determined by e s t i m a t i n g t h e a b i l i t y of c e l l s g i v e n a h y p o t o n i c shock t o t h e n reduce t h e i r c e l l volume (as measured by a C o u l t e r Counter C h a n n e l y s e r ) , a p r o c e s s dependent upon t h e a c t i v i t y o f Na+K+ ATPase (Shank and Smith, 1 9 7 6 ) . N i t r o g e n mustard (lo-',) c o m p l e t e l y i n h i b i t e d t h e r e c o v e r y from hypotonic shock and t h i s c o n c e n t r a t i o n c o r r e s p o n d s t o t h a t which, under i d e n t i c a l i n c u b a t i o n c o n d i t i o n s , caused >90% The monofunctional analogue (11) w a s i n a c t i v e a t lo-% cell k i l l i n t h e bioassay. I t i s concluded t h a t t h e i n h i b i t i o n o f c e l l membrane ATPases by n i t r o g e n mustard may be an i m p o r t a n t f a c e t o f i t s mechanism o f antitumour a c t i o n .
Connors, T.A. ( 1 9 7 1 ) i n Fundamentals o f Biochemical Pharmacology ( e d . Z.M. p461 Pergamon P r e s s , Oxford. Dornand, J. e t a1 (1978) Biochim. Biophys. Acta, 509: 194-200 Kasorov, L . B . and Friedman, H . (1974) Cancer Res., 34: 1862-1865 S a n u i , H . and Rubin, A.H. ( 1 9 7 8 ) , J . C e l l P h y s i o l . 96: 265-278 ( 1 9 7 6 ) , J . C e l l P h y s i o l . 87: 377-387 Shank, B.B. and Smith, N.E. Skou, J.C.(1963) , Biochem. Biophys. R e s . Commun. 10: 79-84. Sweadner, K . J . (1977) Biochem. Biophys. Res. Corn-un. 78: 962-969 Wheeler, G . P . (1967) Fed. Proc. 26: 885-892.
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