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Nonchelational cell growth inhibition by EDTA
Life Sciences, Vol. 39, pp. 1787-1793 Printed in the U.S.A.
NONCHELATIONAL
Pergamon Journal
CELL GROWTH
Philip
INHIBITION
BY EDTA
Skehan
The O n c o l o g y R e s e a r c h Group, D e p a r t m e n t of P h a r m a c o l o g y , F a c u l t y of M e d i c i n e , The U n i v e r s i t y of Calgary, Calgary, A l b e r t a , C a n a d a T 2 N 4NI. (Received in final form August ii, 1986) SUMMARY The a b i l i t y of the d i n i t r i l o t e t r a a c e t a t e s EDTA, CDTA, and E G T A to i n h i b i t the g r o w t h of rat C6 g l i o m a cells was not p r o p o r t i o n a l to t h e i r c h e l a t i o n a l s t a b i l i t y c o n s t a n t s , s u g g e s t i n g a n o n c h e l a t i o n a l m e c h a n i s m of action. Ion a n t a g o n i z a t i o n studies s u p p o r t e d this hypothesis. G r o w t h i n h i b i t i o n did not a p p e a r to be c a u s e d by an E D T A - i o n c o o r d i n a t i o n complex. The c h e m i c a l s t r u c t u r e of the DNTAs s u g g e s t s that t h e y m i g h t act by: (i) i h c r e a s i n g zeta p o t e n t i a l e l e c t r o n e g a t i v i t y and a l t e r i n g local p h y s i c a l p r o p e r t i e s ; (2) m a c r o m o l e c u l a r c r o s s l i n k i n g ; and (3) d i s r u p t i n g h y d r o g e n bonds and h y d r o p h o b i c i n t e r a c t i o n s . Intere s t i n g l y , t h e i r a b i l i t y to inhibit g r o w t h p a r a l l e l e d t h e i r h y d r o p h o b i c s u r f a c e areas. Ethylenediaminetetraacetate (EDTA) is a p o t e n t i n h i b i t o r of c e l l u l a r p r o l i f e r a t i o n and D N A s y n t h e s i s in m a m m a l i a n cells (1-4). B e c a u s e these e f f e c t s are a n t a g o n i z e d by the m u l t i v a l e n t p h y s i o l o g i c a l c a t i o n s Ca, Mg, and Zn (1-3), it is g e n e r a l l y a s s u m e d that E D T A i n h i b i t s g r o w t h c h e l a t i o n a l l y by c a u s i n g the d e s o r p t i o n of these ions from c a t a l y t i c a l l y i m p o r t a n t c o o r d i n a t i o n sites of one or m o r e m e t a l l o p r o t e i n s . However, d i n i t r i l o t e t r a a c e t a t e s such as E D T A are not s p e c i f i c for Ca, Mg, or Zn, (5), have m u c h h i g h e r a f f i n i t i e s for m o s t o t h e r b i o l o g i c a l p o l y v a l e n t c a t i o n s and trace m e t a l s (5), and f r e q u e n t l y i n h i b i t g r o w t h at c o n c e n t r a t i o n s too low to s i g n i f i c a n t l y alter free ion c o n c e n t r a t i o n s (2,3,4,6,7). In addition, the a b i l i t y of c a t i o n s to a n t a g o n i z e E D T A g r o w t h i n h i b i t i o n does not f o l l o w the a c t i v i t y s e q u e n c e e x p e c t e d for a c h e l a t i o n a l p r o c e s s in p h y t o h e m a g g l u t i n i n s t i m u l a t e d l y m p h o c y t e s (2) and chick e m b r y o cells (3). Thus it is not c l e a r that E D T A i n h i b i t s growth by a c h e l a t i o n a l m e c h a n i s m (4), nor has it been e s t a b l i s h e d that it acts t h r o u g h its e f f e c t s on Ca, Mg, or Zn (2,4). The p r e s e n t r e p o r t p r o v i d e s a d d i t i o n a l e v i d e n c e that E D T A i n h i b i t s g r o w t h by a n o n c h e l a t i o n a l m e c h a n i s m of action. Materials
and M e t h o d s
Rat C6 glioma cells w e r e o b t a i n e d from the A m e r i c a n Type Culture Collection. S t o c k s w e r e m a i n t a i n e d in T75 flasks 0024-3205/86 $3.00 + .00 Copyright (c) 1986 Pergamon Journals Ltd.
1788
Nonchelational Growth Inhibition by EDTA
Vol. 39, No. 19, 1986
z 188
x
58
g8 J -58
u z-188 18
18
18
18
18
10
18
18
MOLRR CONCENTRRTION
F I G U R E i. D o s e - r e s p o n s e c u r v e s for the e f f e c t s of D N T A s on C6 g r o w t h and c y t o t o x i c i t y . G r o w t h s t i m u l a t i o n is r e f l e c t e d by r e s p o n s e v a l u e s g r e a t e r t h a n 100%, i n h i b i t i o n by p o s i t i v e v a l u e s of l e s s t h a n 100%, and c y t o t o x i c i t y by n e g a t i v e v a l u e s . Cells w e r e i n c u b a t e d w i t h the d r u g s for 48 h o u r s in g r o w t h m e d i u m . c o n t a i n i n g 80 ml of g r o w t h m e d i u m c h a n g e d at 48 h o u r i n t e r v a l s . G r o w t h m e d i u m c o n s i s t e d of b i c a r b o n a t e b u f f e r e d a u t o c l a v a b l e E a g l e ' s M E M s u p p l e m e n t e d w i t h g l u t a m i n e and 5% f e t a l c a l f serum. E x p e r i m e n t a l c u l t u r e s w e r e p l a t e d at 2 0 0 , 0 0 0 c e l l s p e r 35 m m t i s s u e c u l t u r e d i s h in 2.5 ml of g r o w t h m e d i u m . F o l l o w i n g a 2-3 h o u r c e l l a t t a c h m e n t p e r i o d , m e d i u m w a s r e p l a c e d w i t h 3 ml of t e s t s o l u t i o n d i s s o l v e d in f r e s h g r o w t h m e d i u m and c u l t u r e s i n c u b a t e d for an a d d i t i o n a l 48 h o u r s . Cell protein content was u s e d as a g r o w t h i n d e x . Cultures were washed 4 times with buffer and f i x e d w i t h c o l d 5% TCA. Culture protein content was measured by the m e t h o d of O y a m a a n d E a g l e (8). Background values, o b t a i n e d f r o m b l a n k w e l l s l a c k i n g c e l l s but i n c u b a t e d w i t h m e d i u m , w e r e s u b t r a c t e d f r o m all s a m p l e s p r i o r to f u r t h e r calculation. D r u g i n d u c e d g r o w t h i n h i b i t i o n as a p e r c e n t of c o n t r o l g r o w t h w a s c a l c u l a t e d as 100 x (Pd - P o ) / ( P c - Po), w h e r e Pd and Pc are r e s p e c t i v e l y the cell p r o t e i n c o n t e n t s of d r u g t r e a t e d and c o n t r o l s a m p l e s at the end of an a s s a y and Po is the c e l l p r o t e i n c o n t e n t at t i m e z e r o w h e n d r u g s w e r e a d d e d to the cultures. C y t o t o x i c i t y w a s e x p r e s s e d as a p e r c e n t of the t i m e z e r o c u l t u r e p r o t e i n c o n t e n t and c a l c u l a t e d as I00 x (Po Pd)/Po. I n d i c e s u s e d to a s s e s s d r u g a c t i v i t y w e r e the IC50 ( c o n c e n t r a t i o n p r o d u c i n g 50% i n h i b i t i o n of net g r o w t h ) and L C 5 0 ( c o n c e n t r a t i o n p r o d u c i n g 50% net l o s s of c e l l p r o t e i n ) . The indices were determined graphically from dose-response curves computer-fitted using a cubic spline algorithm. Percent a n t a g o n i z a t i o n by an ion of a c h e l a t o r ' s i n h i b i t i o n of g r o w t h w a s c a l c u l a t e d as i00 x [IChel - ( I ) ( C h e l ) / C o n ] / [ I - ( I ) ( C h e l ) / C o n ] , w h e r e I, Chel, IChel, and Con are the c e l l u l a r p r o t e i n c o n t e n t s of c u l t u r e s t r e a t e d w i t h a n t a g o n i z i n g ion, c h e l a t o r , ion p l u s c h e l a t o r , and c o n t r o l m e d i u m r e s p e c t i v e l y . The t e r m ( I ) ( C h e l ) / C o n r e p r e s e n t s the v a l u e e x p e c t e d f o r an ion w h i c h
Vol. 39, No. 19, 1986
Nonchelational Growth Inhibition by EDTA
TABLE
1789
1
D i n i t r i l o t e t r a a c e t a t e E f f e c t s On C6 G r o w t h I n h i b i t i o n And C y t o t o x i c i t y
Compound CDTA EDTA EGTA
Millimolar IC50
Concentration LC50
0.27 0.10 2.8
1.6 2.9 7.4
e x e r t e d zero a n t a g o n i z a t i o n of a c h e l a t o r ' s effect. E D T A and a n t a g o n i z i n g ions w e r e p r e - m i x e d in g r o w t h m e d i u m b e f o r e a d d i t i o n to test cultures. A n t a g o n i z a t i o n data are p r e s e n t e d for ion c o n c e n t r a t i o n s up to the h i g h e s t v a l u e w h i c h did not itself produce cytotoxicity. EDTA, e t h y l e n e g l y c o l - b i s - ( B - a m i n o e t h y l e t h e r ) N - N ' t e t r a a c e t i c acid (EGTA), and t r a n s - l , 2 - d i a m i n o c y c l o h e x a n e N , N , N ' , N ' - t e t r a a c e t i c acid (CDTA) w e r e o b t a i n e d from Sigma C h e m i c a l Company. RESULTS The d i n i t r i l o t e t r a a c e t a t e s EDTA, CDTA, and E G T A e x e r t e d a t r i p h a s i c e f f e c t upon rat C6 glioma c u l t u r e s (Fig.l, Table i). L o w c o n c e n t r a t i o n s p r o m o t e d g r o w t h s l i g h t l y w i t h an a c t i v i t y s e q u e n c e of E G T A > C D T A = EDTA. Moderate concentrations were c y t o s t a t i c . The a c t i v i t y s e q u e n c e for g r o w t h i n h i b i t i o n was E D T A > C D T A > E G T A w i t h IC50 v a l u e s of i00 uM, 270 uM, and 2.8 m M respectively. High c o n c e n t r a t i o n s w e r e c y t o t o x i c w i t h an a c t i v i t y s e q u e n c e of C D T A > E D T A > E G T A and c o r r e s p o n d i n g LC50 v a l u e s of 1.6, 2.9, and 7.4 mM. F i g u r e 2 i l l u s t r a t e s the a b i l i t y of a v a r i e t y of m u l t i v a l e n t i n o r g a n i c c a t i o n s to a n t a g o n i z e the C6 g r o w t h i n h i b i t i o n caused by 1 m M EDTA. The d o s e - r e s p o n s e curves w e r e c a r r i e d out in i0 fold c o n c e n t r a t i o n steps up to the h i g h e s t level of an ion that did not p r o d u c e overt c y t o t o x i c i t y . A b i l i t y to a n t a g o n i z e E D T A g r o w t h i n h i b i t i o n f o l l o w e d the s e q u e n c e Mo > Zn > A1 > Fe > Mn > Co > Ca => Cu >= 0 = Mg. DISCUSSION E D T A is a p o t e n t i n h i b i t o r of cell g r o w t h w h o s e e f f e c t s are a n t a g o n i z e d by the p h y s i o l o g i c a l cations Ca, Mg, and Zn (2,3,6,7). It is g e n e r a l l y a s s u m e d that E D T A i n h i b i t s g r o w t h c h e l a t i o n a l l y by c o m p e t e t i v e l y d e s o r b i n g one or m o r e of these ions from c a t a l y t i c a l l y i m p o r t a n t m e t a l l o p r o t e i n c o o r d i n a t i o n sites. However, d i n i t r i l o t e t r a a c e t a t e s (DNTA) such as E D T A are o f t e n e f f e c t i v e g r o w t h i n h i b i t o r s at v e r y low c o n c e n t r a t i o n s that c a n n o t s i g n i f i c a n t l y a l t e r free ion c o n c e n t r a t i o n s (2,3,6,7). The DNTAs do not n e c e s s a r i l y r e d u c e the c o n t e n t of Ca, Mg, and Zn ions in b i o l o g i c a l systems, and can s o m e t i m e s a c t u a l l y e l e v a t e them (9-11). In a d d i t i o n , the DNTAs can p r o d u c e large i n t r a c e l l u l a r c o n c e n t r a t i o n changes in b i o l o g i c a l l y i m p o r t a n t c a t i o n s such as Na and K, for w h i c h t h e y have little a f f i n i t y (9-11). These are p r e s u m a b l y s e c o n d a r y e f f e c t s w h i c h result from
1790
Nonchelational Growth Inhibition by EDTA
I
I
I
]
I
I
I
60
J
I
I
6@
68
Zn
Z
Vol. 39, No. 19, 1986
Mn
0
50
50
50
C ~ Z
o
NO
40
40
~7
40
e C~
~o
Z W U
2~
20
[o
1@
~0
u
io
Mg -1@ I_~
I_@ <
l-6
I- 5
I_ 4
HOLRR CONCENTRRTION
~
_
MOLRR CONCENTRRTION
0
.
.
.
.
Z --
MOLRR CONCENTRRTION
FIGURE 2 . I o n i c antagonization o f C6 g r o w t h inhibition b y EDTA. Cells were incubated with solutions for 48 h o u r s i n c o m p l e t e growth medium. e l e c t r o c h e m i c a l c o u p l i n g b e t w e e n d i f f e r e n t i o n i c s p e c i e s (12). The D N T A c h e l a t o r s a r e not s p e c i f i c for Ca, Mg, or Zn (Table 2). T h e y c o m p l e x w i t h v i r t u a l l y all b i o l o g i c a l l y i m p o r t a n t m u l t i v a l e n t c a t i o n s , i n c l u d i n g Co, Cu, Fe, Mn, and Ni, and t h e i r a f f i n i t i e s for Ca and M g a r e c o m p a r a t i v e l y low. S i n c e the c h e l a t i o n a l s t a b i l i t y c o n s t a n t is a l o g a r i t h m i c f u n c t i o n , a u n i t d i f f e r e n c e in it r e p r e s e n t s a i0 f o l d d i f f e r e n c e in a c t u a l a f f i n i t y for an ion (5). The a f f i n i t y of E D T A f o r Cu and Ni is 100 t i m e s g r e a t e r t h a n for Zn and I0 m i l l i o n t i m e s g r e a t e r t h a n for Ca (Table 2). In a p u r e c h e l a t i o n a l p r o c e s s , c o m p e t i n g i o n s are a n t a g o n i s t i c at s t o i c h i o m e t r i c c o n c e n t r a t i o n s , and the o r d e r of t h e i r e f f e c t i v e n e s s is p r o p o r t i o n a l to the s t a b i l i t y c o n s t a n t s of t h e i r c o m p l e x e s w i t h the c h e l a t o r (5). The m o d u l a t i o n of C6 g r o w t h by E D T A did not e x h i b i t t h e s e c h a r a c t e r i s t i c s . The a c t i v i t y of t h e D N T A s w a s E G T A > C D T A = E D T A for g r o w t h s t i m u l a t i o n and E D T A > C D T A > E G T A for g r o w t h i n h i b i t i o n (Fig. i). T h e s e s e q u e n c e s do n o t p a r a l l e l the c h e l a t i o n a l s t a b i l i t y c o n s t a n t s of the D N T A s , w h i c h f o l l o w the o r d e r of C D T A > E G T A > E D T A for Ca and C D T A > E D T A > E G T A for the r e m a i n i n g p h y s i o l o g i c a l c a t i o n s and t r a c e e l e m e n t s s h o w n in T a b l e 2. By c o n t r a s t , D N T A c y t o t o x i c i t y d i s p l a y e d an a c t i v i t y s e q u e n c e of C D T A > E D T A > EGTA, the p a t t e r n e x p e c t e d for a c h e l a t i o n a l m e c h a n i s m i n v o l v i n g a n y o f the ions l i s t e d in T a b l e 2 e x c e p t Ca. T h u s w h i l e the c y t o t o x i c i t y of E D T A m a y i n v o l v e a c h e l a t i o n a l m e c h a n i s m of a c t i o n , its s t i m u l a t i o n and i n h i b i t i o n of g r o w t h a p p a r e n t l y do not.
Vol. 39, No. 19, 1986
Nonchelational Growth Inhibition by EDTA
1791
F u r t h e r e v i d e n c e a g a i n s t the c h e l a t i o n a l h y p o t h e s i s of E D T N g r o w t h i n h i b i t i o n c o m e s f r o m the f i n d i n g t h a t t h e a b i l i t y of e x o g e n o u s c a t i o n s to a n t a g o n i z e the e f f e c t w a s not p r o p o r t i o n a l to t h e s t a b i l i t y c o n s t a n t s of t h e i r E D T N c h e l a t e s . The a c t i v i t y s e q u e n c e e x p e c t e d for a n t a g o n i z a t i o n of a e h e l a t i o n a l p r o c e s s is Cu > Co > Zn > F e ( I I ) > Mn > Ca > M g (Table 2). N very different s e q u e n c e w a s o b s e r v e d f o r r e v e r s a l of E D T N g r o w t h i n h i b i t i o n : M o > Zn > Nl > F e ( I I ) > Mn > Co > Ca >= Cu >= M g = 0 (Figs. 2). Cu, w h i c h s h o u l d h a v e b e e n t h e b e s t a n t a g o n i s t , had v i r t u a l l y no e f f e c t o v e r the o b s e r v a b l e c o n c e n t r a t i o n r a n g e . A l t h o u g h the a f f i n i t y of E D T N for Ca is i00 m i l l i o n t i m e s l e s s t h a n for Cu
TABLE Chelator Compound CDTA EDTA EGTA * Values
2
Stability
Cu
Ni
Co
Zn
21.9 18.7 17.6
20.2 18.5 13.5
19.6 16.3
19.4 16.4 12.2
from Martell
and
Smith,
Constants Fe(II) 18.9 14.3 11.8
Mn
Ca
Mg
17.4 13.8 12.2
13.2 10.6 10.9
ii.i 8.8 5.3
1982.
( T a b l e 2), Ca w a s s i g n i f i c a n t l y b e t t e r as an a n t a g o n i s t of E D T A growth inhibition. B e c a u s e all of the c a t i o n s e x a m i n e d in the reversal experiments have relatively high EDTA stability c o n s t a n t s , t h e y s h o u l d all be s t o i c h i o m e t r i c a l l y a n t a g o n i s t i c o v e r n e a r l y i d e n t i c a l c o n c e n t r a t i o n r a n g e s if E D T A i n h i b i t s g r o w t h by c a u s i n g t h e d i s s o c i a t i o n of a c a t i o n f r o m a c e l l u l a r c o o r d i n a t i o n site. S u c h b e h a v i o r w a s not o b s e r v e d . In m e d i u m c o n t a i n i n g 1 . 3 6 m M Ca and 0.81 m M Mg, the g r o w t h i n h i b i t i o n produced by 1 mM EDTA was antagonized by submicromolar c o n c e n t r a t i o n s of Mo, m i c r o m o l a r c o n c e n t r a t i o n s of Zn, c i r c a i0 m i c r o m o l a r c o n c e n t r a t i o n s of AI, Fe, and Mn, s u b m i l l i m o l a r c o n c e n t r a t i o n s of Co, and m i l l i m o l a r Ca c o n c e n t r a t i o n s . Mg was i n e f f e c t i v e as an a n t a g o n i s t at c o n c e n t r a t i o n s of up to 10 f o l d molar excess. W i d e v a r i a t i o n s in the e f f e c t i v e c o n c e n t r a t i o n s of a n t a g o n i z i n g i o n s h a v e a l s o b e e n r e p o r t e d b y R u b i n (3) for the Ca, Mg, and Zn r e v e r s a l of E D T A i n h i b i t i o n of t h y m i d i n e incorporation by chick embryo cells. T h e s e o b s e r v a t i o n s are not c o m p a t i b l e w i t h a c h e l a t i o n a l m e c h a n i s m of a c t i o n . The c o o r d i n a t i o n c o m p l e x e s w h i c h E D T A f o r m s w i t h c a t i o n s can themselves exert direct biological effects which are independent of and d i s t i n c t f r o m c h e l a t i o n a l t i t r a t i o n ( 1 3 , 1 4 ) . Were this the m e c h a n i s m of C6 g r o w t h i n h i b i t i o n by E D T A , t h e n a d d i t i o n of t h e a p p r o p r i a t e e x o g e n o u s c a t i o n , at c o n c e n t r a t i o n s w h e r e the ion i t s e l f is n o t t o x i c , s h o u l d p o t e n t i a t e the e f f e c t of EDTA. P o t e n t i a t i o n w a s n o t o b s e r v e d w i t h a n y of the c a t i o n s e x a m i n e d (Fig. 2). T h i s s u g g e s t s e i t h e r t h a t the a c t i v e c o o r d i n a t i o n c o m p l e x i n v o l v e s a s p e c i e s of ion t h a t w a s not t e s t e d , or t h a t c o o r d i n a t i o n c o m p l e x e s are not r e s p o n s i b l e f o r C6 g r o w t h inhibition.
1792
Nonchelational Growth Inhibition by EDTA
Vol. 39, No. 19, 1986
The c h e m i c a l s t r u c t u r e of the D N T A s s u g g e s t s three p o s s i b l e m e c h a n i s m s of a c t i o n o t h e r than ion c h e l a t i o n : (i) the e l e c t r o s t a t i c i n t e r a c t i o n of t h e i r c a r b o x y l g r o u p s w i t h the p o s i t i v e fixed c h a r g e s of b i o l o g i c a l s u r f a c e s and m a c r o m o l e c u l e s can be e x p e c t e d to i n c r e a s e zeta p o t e n t i a l e l e c t r o n e g a t i v i t y and m o d i f y i n t e r f a c i a l p r o p e r t i e s such as local ion c o n c e n t r a t i o n s , d i e l e c t r i c s u s c e p t i b i l i t y , and s o l v e n t s t r u c t u r i n g ; (2) by v i r t u e of t h e i r m u l t i p l e c a r b o x y l r e s i d u e s the D N T A s p o s s e s s the c a p a c i t y to serve as m a c r o m o l e c u l a r c r o s s l i n k i n g agents, and could p o t e n t i a l l y a l t e r c y t o p l a s m i c c o l l o i d p h a s e c h a r a c t e r i s t i c s ; (3) t h e i r n i t r i l o a c e t a t e r e s i d u e s are s t r u c t u r a l l y s i m i l a r to s e v e r a l c l a s s e s of d e n a t u r i n g s o l v e n t s w h i c h d i s r u p t h y d r o g e n b o n d s and, d e p e n d i n g on c o n c e n t r a t i o n , can e i t h e r s t a b i l i z e or d i s r u p t h y d r o p h o b i c i n t e r a c t i o n s (15-17). An u n r e l a t e d chelator, 1 , 1 0 - p h e n a n t h r o l i n e , has b e e n shown to i n h i b i t y e a s t a l c o h o l d e h y d r o g e n a s e by a h y d r o p h o b i c r a t h e r than a c h e l a t i o n a l m e c h a n i s m of a c t i o n (18). It has been s u g g e s t e d that its i n h i b i t i o n of DNA s y n t h e s i s m a y a l s o i n v o l v e a h y d r o p h o b i c r a t h e r than a c h e l a t i o n a l m e c h a n i s m (19). The a b i l i t y of the D N T A s to i n h i b i t C6 g r o w t h (Fig. i) p a r a l l e l e d t h e i r h y d r o p h o b i c s u r f a c e areas. Thus the p o s s i b i l i t y n e e d s to be c o n s i d e r e d that a n u m b e r of chelators, i n c l u d i n g EDTA, m a y exert some of t h e i r b i o l o g i c a l e f f e c t s t h r o u g h a h y d r o p h o b i c r a t h e r than a c h e l a t i o n a l m e c h a n i s m of action. Finally, E D T A i n h i b i t s the g r o w t h of E h r l i c h a s c i t e s cells w i t h o u t s i g n i f i c a n t i n t r a c e l l u l a r a c c u m u l a t i o n (4). This r a i s e s the p o s s i b i l i t y that it m a y act at e i t h e r the e x t r a c e l l u l a r face of the p l a s m a m e m b r a n e or the e x t e r n a l s u r f a c e coat. ACKNOWLEDGEMENTS The a u t h o r w i s h e s to t h a n k Ping C h i u for her e x c e l l e n t technical assistance. This w o r k was s u p p o r t e d by g r a n t s f r o m the A l b e r t a C a n c e r Board and the M e d i c a l R e s e a r c h C o u n c i l of Canada. REFERENCES i. I. L I E B E R M A N and P. OVE, J. Biol. Chem. 237 1 6 3 4 - 1 6 4 2 (1962). 2. R.H. ALFORD, J. Immunol. 104 698-703 (1970). 3. H. RUBIN. Proc. Natl. Acad. Sci. (U.S.) 69 7 1 2 - 7 1 6 (1972). 4. C. K R I S H N A M U R T I , L.A. SARYAN, and D.H. P E T E R I N G , C a n c e r R e s e a r c h 40 4 0 9 2 - 4 0 9 9 (1980). 5. A.E. M A R T E L L and R.M. SMITH, C r i t i c a l S t a b i l i t y C o n s t a n t s . P l e n u m Press, New Y o r k (1982). 6. M. F U J I O K A and I. L I E B E R M A N , J. Biol. Chem. 239 1 1 6 4 - 1 1 6 7 (1964). 7. J.K. C H E S T E R S , Biochem. J. 150 2 1 1 - 2 1 8 (1975). 8. V.I. O Y A M A and H. EAGLE, Proc. Soc. Exp. Biol. Med. 91 305-307 (1956). 9. H. SANUI and N. PACE, J. Cell. Physiol. 69 3-10 (1967). 10. H. SANUI and N. PACE, J. Cell. Physiol. 69 11-20 (1967). ii. D. M O S C A T E L L I , H. SANUI and A.H. RUBIN, J. Cell. Physiol. i01 1 1 7 - 1 2 8 (1979). 12. I.L CAMERON, N.K.R. SMITH, and P. SKEHAN, In Ions and Cell P r o l i f e r a t i o n (Boynton, A.L., M c K E E H A N , W.L., and W H I T F I E L D , J.F. eds.) pp 67-133. A c a d e m i c Press, N e w Y o r k (1982). 13. V. D ' A U R O R A , A.M. STERN, and D.S SIGMAN, Biophys. Res. Comm. 80 1 0 2 5 - 1 0 3 2 (1978).
Vol. 39, No. 19, 1986
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M. TIEN, L.A. M O R E H O U S E , J.R BUCHER, and S.D. AUST, Arch. B i o c h e m . Biophys. 218 4 5 0 - 4 5 8 (1982). M.F. E M E R S O N and A. H O L T Z E R , J. Phys. Chem. 71 3 3 2 0 - 3 3 3 0 (1967). T.T. H E R S K O V I T S , B. G A D E G B E K U , and H. JAILLET, J. Biol. Chem. 245 2 5 8 8 - 2 5 9 8 (1970). L.S. K A M I N S K Y , R.L. WRIGHT, and A.J. DAVISON, B i o c h e m i s t r y i0 4 5 8 - 4 6 2 (1971). B.M. A N D E R S O N , M.L. R E Y N O L D S , and C.D. A N D E R S O N , Biochim. B i o p h y s . A c t a 113 2 3 5 - 2 4 3 (1966). C-H. C H A N G and J. Yarbro, Life S c i e n c e s 22 1 0 0 7 - 1 0 1 0 (1978).
NONCHELATIONAL
Pergamon Journal
CELL GROWTH
Philip
INHIBITION
BY EDTA
Skehan
The O n c o l o g y R e s e a r c h Group, D e p a r t m e n t of P h a r m a c o l o g y , F a c u l t y of M e d i c i n e , The U n i v e r s i t y of Calgary, Calgary, A l b e r t a , C a n a d a T 2 N 4NI. (Received in final form August ii, 1986) SUMMARY The a b i l i t y of the d i n i t r i l o t e t r a a c e t a t e s EDTA, CDTA, and E G T A to i n h i b i t the g r o w t h of rat C6 g l i o m a cells was not p r o p o r t i o n a l to t h e i r c h e l a t i o n a l s t a b i l i t y c o n s t a n t s , s u g g e s t i n g a n o n c h e l a t i o n a l m e c h a n i s m of action. Ion a n t a g o n i z a t i o n studies s u p p o r t e d this hypothesis. G r o w t h i n h i b i t i o n did not a p p e a r to be c a u s e d by an E D T A - i o n c o o r d i n a t i o n complex. The c h e m i c a l s t r u c t u r e of the DNTAs s u g g e s t s that t h e y m i g h t act by: (i) i h c r e a s i n g zeta p o t e n t i a l e l e c t r o n e g a t i v i t y and a l t e r i n g local p h y s i c a l p r o p e r t i e s ; (2) m a c r o m o l e c u l a r c r o s s l i n k i n g ; and (3) d i s r u p t i n g h y d r o g e n bonds and h y d r o p h o b i c i n t e r a c t i o n s . Intere s t i n g l y , t h e i r a b i l i t y to inhibit g r o w t h p a r a l l e l e d t h e i r h y d r o p h o b i c s u r f a c e areas. Ethylenediaminetetraacetate (EDTA) is a p o t e n t i n h i b i t o r of c e l l u l a r p r o l i f e r a t i o n and D N A s y n t h e s i s in m a m m a l i a n cells (1-4). B e c a u s e these e f f e c t s are a n t a g o n i z e d by the m u l t i v a l e n t p h y s i o l o g i c a l c a t i o n s Ca, Mg, and Zn (1-3), it is g e n e r a l l y a s s u m e d that E D T A i n h i b i t s g r o w t h c h e l a t i o n a l l y by c a u s i n g the d e s o r p t i o n of these ions from c a t a l y t i c a l l y i m p o r t a n t c o o r d i n a t i o n sites of one or m o r e m e t a l l o p r o t e i n s . However, d i n i t r i l o t e t r a a c e t a t e s such as E D T A are not s p e c i f i c for Ca, Mg, or Zn, (5), have m u c h h i g h e r a f f i n i t i e s for m o s t o t h e r b i o l o g i c a l p o l y v a l e n t c a t i o n s and trace m e t a l s (5), and f r e q u e n t l y i n h i b i t g r o w t h at c o n c e n t r a t i o n s too low to s i g n i f i c a n t l y alter free ion c o n c e n t r a t i o n s (2,3,4,6,7). In addition, the a b i l i t y of c a t i o n s to a n t a g o n i z e E D T A g r o w t h i n h i b i t i o n does not f o l l o w the a c t i v i t y s e q u e n c e e x p e c t e d for a c h e l a t i o n a l p r o c e s s in p h y t o h e m a g g l u t i n i n s t i m u l a t e d l y m p h o c y t e s (2) and chick e m b r y o cells (3). Thus it is not c l e a r that E D T A i n h i b i t s growth by a c h e l a t i o n a l m e c h a n i s m (4), nor has it been e s t a b l i s h e d that it acts t h r o u g h its e f f e c t s on Ca, Mg, or Zn (2,4). The p r e s e n t r e p o r t p r o v i d e s a d d i t i o n a l e v i d e n c e that E D T A i n h i b i t s g r o w t h by a n o n c h e l a t i o n a l m e c h a n i s m of action. Materials
and M e t h o d s
Rat C6 glioma cells w e r e o b t a i n e d from the A m e r i c a n Type Culture Collection. S t o c k s w e r e m a i n t a i n e d in T75 flasks 0024-3205/86 $3.00 + .00 Copyright (c) 1986 Pergamon Journals Ltd.
1788
Nonchelational Growth Inhibition by EDTA
Vol. 39, No. 19, 1986
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F I G U R E i. D o s e - r e s p o n s e c u r v e s for the e f f e c t s of D N T A s on C6 g r o w t h and c y t o t o x i c i t y . G r o w t h s t i m u l a t i o n is r e f l e c t e d by r e s p o n s e v a l u e s g r e a t e r t h a n 100%, i n h i b i t i o n by p o s i t i v e v a l u e s of l e s s t h a n 100%, and c y t o t o x i c i t y by n e g a t i v e v a l u e s . Cells w e r e i n c u b a t e d w i t h the d r u g s for 48 h o u r s in g r o w t h m e d i u m . c o n t a i n i n g 80 ml of g r o w t h m e d i u m c h a n g e d at 48 h o u r i n t e r v a l s . G r o w t h m e d i u m c o n s i s t e d of b i c a r b o n a t e b u f f e r e d a u t o c l a v a b l e E a g l e ' s M E M s u p p l e m e n t e d w i t h g l u t a m i n e and 5% f e t a l c a l f serum. E x p e r i m e n t a l c u l t u r e s w e r e p l a t e d at 2 0 0 , 0 0 0 c e l l s p e r 35 m m t i s s u e c u l t u r e d i s h in 2.5 ml of g r o w t h m e d i u m . F o l l o w i n g a 2-3 h o u r c e l l a t t a c h m e n t p e r i o d , m e d i u m w a s r e p l a c e d w i t h 3 ml of t e s t s o l u t i o n d i s s o l v e d in f r e s h g r o w t h m e d i u m and c u l t u r e s i n c u b a t e d for an a d d i t i o n a l 48 h o u r s . Cell protein content was u s e d as a g r o w t h i n d e x . Cultures were washed 4 times with buffer and f i x e d w i t h c o l d 5% TCA. Culture protein content was measured by the m e t h o d of O y a m a a n d E a g l e (8). Background values, o b t a i n e d f r o m b l a n k w e l l s l a c k i n g c e l l s but i n c u b a t e d w i t h m e d i u m , w e r e s u b t r a c t e d f r o m all s a m p l e s p r i o r to f u r t h e r calculation. D r u g i n d u c e d g r o w t h i n h i b i t i o n as a p e r c e n t of c o n t r o l g r o w t h w a s c a l c u l a t e d as 100 x (Pd - P o ) / ( P c - Po), w h e r e Pd and Pc are r e s p e c t i v e l y the cell p r o t e i n c o n t e n t s of d r u g t r e a t e d and c o n t r o l s a m p l e s at the end of an a s s a y and Po is the c e l l p r o t e i n c o n t e n t at t i m e z e r o w h e n d r u g s w e r e a d d e d to the cultures. C y t o t o x i c i t y w a s e x p r e s s e d as a p e r c e n t of the t i m e z e r o c u l t u r e p r o t e i n c o n t e n t and c a l c u l a t e d as I00 x (Po Pd)/Po. I n d i c e s u s e d to a s s e s s d r u g a c t i v i t y w e r e the IC50 ( c o n c e n t r a t i o n p r o d u c i n g 50% i n h i b i t i o n of net g r o w t h ) and L C 5 0 ( c o n c e n t r a t i o n p r o d u c i n g 50% net l o s s of c e l l p r o t e i n ) . The indices were determined graphically from dose-response curves computer-fitted using a cubic spline algorithm. Percent a n t a g o n i z a t i o n by an ion of a c h e l a t o r ' s i n h i b i t i o n of g r o w t h w a s c a l c u l a t e d as i00 x [IChel - ( I ) ( C h e l ) / C o n ] / [ I - ( I ) ( C h e l ) / C o n ] , w h e r e I, Chel, IChel, and Con are the c e l l u l a r p r o t e i n c o n t e n t s of c u l t u r e s t r e a t e d w i t h a n t a g o n i z i n g ion, c h e l a t o r , ion p l u s c h e l a t o r , and c o n t r o l m e d i u m r e s p e c t i v e l y . The t e r m ( I ) ( C h e l ) / C o n r e p r e s e n t s the v a l u e e x p e c t e d f o r an ion w h i c h
Vol. 39, No. 19, 1986
Nonchelational Growth Inhibition by EDTA
TABLE
1789
1
D i n i t r i l o t e t r a a c e t a t e E f f e c t s On C6 G r o w t h I n h i b i t i o n And C y t o t o x i c i t y
Compound CDTA EDTA EGTA
Millimolar IC50
Concentration LC50
0.27 0.10 2.8
1.6 2.9 7.4
e x e r t e d zero a n t a g o n i z a t i o n of a c h e l a t o r ' s effect. E D T A and a n t a g o n i z i n g ions w e r e p r e - m i x e d in g r o w t h m e d i u m b e f o r e a d d i t i o n to test cultures. A n t a g o n i z a t i o n data are p r e s e n t e d for ion c o n c e n t r a t i o n s up to the h i g h e s t v a l u e w h i c h did not itself produce cytotoxicity. EDTA, e t h y l e n e g l y c o l - b i s - ( B - a m i n o e t h y l e t h e r ) N - N ' t e t r a a c e t i c acid (EGTA), and t r a n s - l , 2 - d i a m i n o c y c l o h e x a n e N , N , N ' , N ' - t e t r a a c e t i c acid (CDTA) w e r e o b t a i n e d from Sigma C h e m i c a l Company. RESULTS The d i n i t r i l o t e t r a a c e t a t e s EDTA, CDTA, and E G T A e x e r t e d a t r i p h a s i c e f f e c t upon rat C6 glioma c u l t u r e s (Fig.l, Table i). L o w c o n c e n t r a t i o n s p r o m o t e d g r o w t h s l i g h t l y w i t h an a c t i v i t y s e q u e n c e of E G T A > C D T A = EDTA. Moderate concentrations were c y t o s t a t i c . The a c t i v i t y s e q u e n c e for g r o w t h i n h i b i t i o n was E D T A > C D T A > E G T A w i t h IC50 v a l u e s of i00 uM, 270 uM, and 2.8 m M respectively. High c o n c e n t r a t i o n s w e r e c y t o t o x i c w i t h an a c t i v i t y s e q u e n c e of C D T A > E D T A > E G T A and c o r r e s p o n d i n g LC50 v a l u e s of 1.6, 2.9, and 7.4 mM. F i g u r e 2 i l l u s t r a t e s the a b i l i t y of a v a r i e t y of m u l t i v a l e n t i n o r g a n i c c a t i o n s to a n t a g o n i z e the C6 g r o w t h i n h i b i t i o n caused by 1 m M EDTA. The d o s e - r e s p o n s e curves w e r e c a r r i e d out in i0 fold c o n c e n t r a t i o n steps up to the h i g h e s t level of an ion that did not p r o d u c e overt c y t o t o x i c i t y . A b i l i t y to a n t a g o n i z e E D T A g r o w t h i n h i b i t i o n f o l l o w e d the s e q u e n c e Mo > Zn > A1 > Fe > Mn > Co > Ca => Cu >= 0 = Mg. DISCUSSION E D T A is a p o t e n t i n h i b i t o r of cell g r o w t h w h o s e e f f e c t s are a n t a g o n i z e d by the p h y s i o l o g i c a l cations Ca, Mg, and Zn (2,3,6,7). It is g e n e r a l l y a s s u m e d that E D T A i n h i b i t s g r o w t h c h e l a t i o n a l l y by c o m p e t e t i v e l y d e s o r b i n g one or m o r e of these ions from c a t a l y t i c a l l y i m p o r t a n t m e t a l l o p r o t e i n c o o r d i n a t i o n sites. However, d i n i t r i l o t e t r a a c e t a t e s (DNTA) such as E D T A are o f t e n e f f e c t i v e g r o w t h i n h i b i t o r s at v e r y low c o n c e n t r a t i o n s that c a n n o t s i g n i f i c a n t l y a l t e r free ion c o n c e n t r a t i o n s (2,3,6,7). The DNTAs do not n e c e s s a r i l y r e d u c e the c o n t e n t of Ca, Mg, and Zn ions in b i o l o g i c a l systems, and can s o m e t i m e s a c t u a l l y e l e v a t e them (9-11). In a d d i t i o n , the DNTAs can p r o d u c e large i n t r a c e l l u l a r c o n c e n t r a t i o n changes in b i o l o g i c a l l y i m p o r t a n t c a t i o n s such as Na and K, for w h i c h t h e y have little a f f i n i t y (9-11). These are p r e s u m a b l y s e c o n d a r y e f f e c t s w h i c h result from
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Z --
MOLRR CONCENTRRTION
FIGURE 2 . I o n i c antagonization o f C6 g r o w t h inhibition b y EDTA. Cells were incubated with solutions for 48 h o u r s i n c o m p l e t e growth medium. e l e c t r o c h e m i c a l c o u p l i n g b e t w e e n d i f f e r e n t i o n i c s p e c i e s (12). The D N T A c h e l a t o r s a r e not s p e c i f i c for Ca, Mg, or Zn (Table 2). T h e y c o m p l e x w i t h v i r t u a l l y all b i o l o g i c a l l y i m p o r t a n t m u l t i v a l e n t c a t i o n s , i n c l u d i n g Co, Cu, Fe, Mn, and Ni, and t h e i r a f f i n i t i e s for Ca and M g a r e c o m p a r a t i v e l y low. S i n c e the c h e l a t i o n a l s t a b i l i t y c o n s t a n t is a l o g a r i t h m i c f u n c t i o n , a u n i t d i f f e r e n c e in it r e p r e s e n t s a i0 f o l d d i f f e r e n c e in a c t u a l a f f i n i t y for an ion (5). The a f f i n i t y of E D T A f o r Cu and Ni is 100 t i m e s g r e a t e r t h a n for Zn and I0 m i l l i o n t i m e s g r e a t e r t h a n for Ca (Table 2). In a p u r e c h e l a t i o n a l p r o c e s s , c o m p e t i n g i o n s are a n t a g o n i s t i c at s t o i c h i o m e t r i c c o n c e n t r a t i o n s , and the o r d e r of t h e i r e f f e c t i v e n e s s is p r o p o r t i o n a l to the s t a b i l i t y c o n s t a n t s of t h e i r c o m p l e x e s w i t h the c h e l a t o r (5). The m o d u l a t i o n of C6 g r o w t h by E D T A did not e x h i b i t t h e s e c h a r a c t e r i s t i c s . The a c t i v i t y of t h e D N T A s w a s E G T A > C D T A = E D T A for g r o w t h s t i m u l a t i o n and E D T A > C D T A > E G T A for g r o w t h i n h i b i t i o n (Fig. i). T h e s e s e q u e n c e s do n o t p a r a l l e l the c h e l a t i o n a l s t a b i l i t y c o n s t a n t s of the D N T A s , w h i c h f o l l o w the o r d e r of C D T A > E G T A > E D T A for Ca and C D T A > E D T A > E G T A for the r e m a i n i n g p h y s i o l o g i c a l c a t i o n s and t r a c e e l e m e n t s s h o w n in T a b l e 2. By c o n t r a s t , D N T A c y t o t o x i c i t y d i s p l a y e d an a c t i v i t y s e q u e n c e of C D T A > E D T A > EGTA, the p a t t e r n e x p e c t e d for a c h e l a t i o n a l m e c h a n i s m i n v o l v i n g a n y o f the ions l i s t e d in T a b l e 2 e x c e p t Ca. T h u s w h i l e the c y t o t o x i c i t y of E D T A m a y i n v o l v e a c h e l a t i o n a l m e c h a n i s m of a c t i o n , its s t i m u l a t i o n and i n h i b i t i o n of g r o w t h a p p a r e n t l y do not.
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1791
F u r t h e r e v i d e n c e a g a i n s t the c h e l a t i o n a l h y p o t h e s i s of E D T N g r o w t h i n h i b i t i o n c o m e s f r o m the f i n d i n g t h a t t h e a b i l i t y of e x o g e n o u s c a t i o n s to a n t a g o n i z e the e f f e c t w a s not p r o p o r t i o n a l to t h e s t a b i l i t y c o n s t a n t s of t h e i r E D T N c h e l a t e s . The a c t i v i t y s e q u e n c e e x p e c t e d for a n t a g o n i z a t i o n of a e h e l a t i o n a l p r o c e s s is Cu > Co > Zn > F e ( I I ) > Mn > Ca > M g (Table 2). N very different s e q u e n c e w a s o b s e r v e d f o r r e v e r s a l of E D T N g r o w t h i n h i b i t i o n : M o > Zn > Nl > F e ( I I ) > Mn > Co > Ca >= Cu >= M g = 0 (Figs. 2). Cu, w h i c h s h o u l d h a v e b e e n t h e b e s t a n t a g o n i s t , had v i r t u a l l y no e f f e c t o v e r the o b s e r v a b l e c o n c e n t r a t i o n r a n g e . A l t h o u g h the a f f i n i t y of E D T N for Ca is i00 m i l l i o n t i m e s l e s s t h a n for Cu
TABLE Chelator Compound CDTA EDTA EGTA * Values
2
Stability
Cu
Ni
Co
Zn
21.9 18.7 17.6
20.2 18.5 13.5
19.6 16.3
19.4 16.4 12.2
from Martell
and
Smith,
Constants Fe(II) 18.9 14.3 11.8
Mn
Ca
Mg
17.4 13.8 12.2
13.2 10.6 10.9
ii.i 8.8 5.3
1982.
( T a b l e 2), Ca w a s s i g n i f i c a n t l y b e t t e r as an a n t a g o n i s t of E D T A growth inhibition. B e c a u s e all of the c a t i o n s e x a m i n e d in the reversal experiments have relatively high EDTA stability c o n s t a n t s , t h e y s h o u l d all be s t o i c h i o m e t r i c a l l y a n t a g o n i s t i c o v e r n e a r l y i d e n t i c a l c o n c e n t r a t i o n r a n g e s if E D T A i n h i b i t s g r o w t h by c a u s i n g t h e d i s s o c i a t i o n of a c a t i o n f r o m a c e l l u l a r c o o r d i n a t i o n site. S u c h b e h a v i o r w a s not o b s e r v e d . In m e d i u m c o n t a i n i n g 1 . 3 6 m M Ca and 0.81 m M Mg, the g r o w t h i n h i b i t i o n produced by 1 mM EDTA was antagonized by submicromolar c o n c e n t r a t i o n s of Mo, m i c r o m o l a r c o n c e n t r a t i o n s of Zn, c i r c a i0 m i c r o m o l a r c o n c e n t r a t i o n s of AI, Fe, and Mn, s u b m i l l i m o l a r c o n c e n t r a t i o n s of Co, and m i l l i m o l a r Ca c o n c e n t r a t i o n s . Mg was i n e f f e c t i v e as an a n t a g o n i s t at c o n c e n t r a t i o n s of up to 10 f o l d molar excess. W i d e v a r i a t i o n s in the e f f e c t i v e c o n c e n t r a t i o n s of a n t a g o n i z i n g i o n s h a v e a l s o b e e n r e p o r t e d b y R u b i n (3) for the Ca, Mg, and Zn r e v e r s a l of E D T A i n h i b i t i o n of t h y m i d i n e incorporation by chick embryo cells. T h e s e o b s e r v a t i o n s are not c o m p a t i b l e w i t h a c h e l a t i o n a l m e c h a n i s m of a c t i o n . The c o o r d i n a t i o n c o m p l e x e s w h i c h E D T A f o r m s w i t h c a t i o n s can themselves exert direct biological effects which are independent of and d i s t i n c t f r o m c h e l a t i o n a l t i t r a t i o n ( 1 3 , 1 4 ) . Were this the m e c h a n i s m of C6 g r o w t h i n h i b i t i o n by E D T A , t h e n a d d i t i o n of t h e a p p r o p r i a t e e x o g e n o u s c a t i o n , at c o n c e n t r a t i o n s w h e r e the ion i t s e l f is n o t t o x i c , s h o u l d p o t e n t i a t e the e f f e c t of EDTA. P o t e n t i a t i o n w a s n o t o b s e r v e d w i t h a n y of the c a t i o n s e x a m i n e d (Fig. 2). T h i s s u g g e s t s e i t h e r t h a t the a c t i v e c o o r d i n a t i o n c o m p l e x i n v o l v e s a s p e c i e s of ion t h a t w a s not t e s t e d , or t h a t c o o r d i n a t i o n c o m p l e x e s are not r e s p o n s i b l e f o r C6 g r o w t h inhibition.
1792
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Vol. 39, No. 19, 1986
The c h e m i c a l s t r u c t u r e of the D N T A s s u g g e s t s three p o s s i b l e m e c h a n i s m s of a c t i o n o t h e r than ion c h e l a t i o n : (i) the e l e c t r o s t a t i c i n t e r a c t i o n of t h e i r c a r b o x y l g r o u p s w i t h the p o s i t i v e fixed c h a r g e s of b i o l o g i c a l s u r f a c e s and m a c r o m o l e c u l e s can be e x p e c t e d to i n c r e a s e zeta p o t e n t i a l e l e c t r o n e g a t i v i t y and m o d i f y i n t e r f a c i a l p r o p e r t i e s such as local ion c o n c e n t r a t i o n s , d i e l e c t r i c s u s c e p t i b i l i t y , and s o l v e n t s t r u c t u r i n g ; (2) by v i r t u e of t h e i r m u l t i p l e c a r b o x y l r e s i d u e s the D N T A s p o s s e s s the c a p a c i t y to serve as m a c r o m o l e c u l a r c r o s s l i n k i n g agents, and could p o t e n t i a l l y a l t e r c y t o p l a s m i c c o l l o i d p h a s e c h a r a c t e r i s t i c s ; (3) t h e i r n i t r i l o a c e t a t e r e s i d u e s are s t r u c t u r a l l y s i m i l a r to s e v e r a l c l a s s e s of d e n a t u r i n g s o l v e n t s w h i c h d i s r u p t h y d r o g e n b o n d s and, d e p e n d i n g on c o n c e n t r a t i o n , can e i t h e r s t a b i l i z e or d i s r u p t h y d r o p h o b i c i n t e r a c t i o n s (15-17). An u n r e l a t e d chelator, 1 , 1 0 - p h e n a n t h r o l i n e , has b e e n shown to i n h i b i t y e a s t a l c o h o l d e h y d r o g e n a s e by a h y d r o p h o b i c r a t h e r than a c h e l a t i o n a l m e c h a n i s m of a c t i o n (18). It has been s u g g e s t e d that its i n h i b i t i o n of DNA s y n t h e s i s m a y a l s o i n v o l v e a h y d r o p h o b i c r a t h e r than a c h e l a t i o n a l m e c h a n i s m (19). The a b i l i t y of the D N T A s to i n h i b i t C6 g r o w t h (Fig. i) p a r a l l e l e d t h e i r h y d r o p h o b i c s u r f a c e areas. Thus the p o s s i b i l i t y n e e d s to be c o n s i d e r e d that a n u m b e r of chelators, i n c l u d i n g EDTA, m a y exert some of t h e i r b i o l o g i c a l e f f e c t s t h r o u g h a h y d r o p h o b i c r a t h e r than a c h e l a t i o n a l m e c h a n i s m of action. Finally, E D T A i n h i b i t s the g r o w t h of E h r l i c h a s c i t e s cells w i t h o u t s i g n i f i c a n t i n t r a c e l l u l a r a c c u m u l a t i o n (4). This r a i s e s the p o s s i b i l i t y that it m a y act at e i t h e r the e x t r a c e l l u l a r face of the p l a s m a m e m b r a n e or the e x t e r n a l s u r f a c e coat. ACKNOWLEDGEMENTS The a u t h o r w i s h e s to t h a n k Ping C h i u for her e x c e l l e n t technical assistance. This w o r k was s u p p o r t e d by g r a n t s f r o m the A l b e r t a C a n c e r Board and the M e d i c a l R e s e a r c h C o u n c i l of Canada. REFERENCES i. I. L I E B E R M A N and P. OVE, J. Biol. Chem. 237 1 6 3 4 - 1 6 4 2 (1962). 2. R.H. ALFORD, J. Immunol. 104 698-703 (1970). 3. H. RUBIN. Proc. Natl. Acad. Sci. (U.S.) 69 7 1 2 - 7 1 6 (1972). 4. C. K R I S H N A M U R T I , L.A. SARYAN, and D.H. P E T E R I N G , C a n c e r R e s e a r c h 40 4 0 9 2 - 4 0 9 9 (1980). 5. A.E. M A R T E L L and R.M. SMITH, C r i t i c a l S t a b i l i t y C o n s t a n t s . P l e n u m Press, New Y o r k (1982). 6. M. F U J I O K A and I. L I E B E R M A N , J. Biol. Chem. 239 1 1 6 4 - 1 1 6 7 (1964). 7. J.K. C H E S T E R S , Biochem. J. 150 2 1 1 - 2 1 8 (1975). 8. V.I. O Y A M A and H. EAGLE, Proc. Soc. Exp. Biol. Med. 91 305-307 (1956). 9. H. SANUI and N. PACE, J. Cell. Physiol. 69 3-10 (1967). 10. H. SANUI and N. PACE, J. Cell. Physiol. 69 11-20 (1967). ii. D. M O S C A T E L L I , H. SANUI and A.H. RUBIN, J. Cell. Physiol. i01 1 1 7 - 1 2 8 (1979). 12. I.L CAMERON, N.K.R. SMITH, and P. SKEHAN, In Ions and Cell P r o l i f e r a t i o n (Boynton, A.L., M c K E E H A N , W.L., and W H I T F I E L D , J.F. eds.) pp 67-133. A c a d e m i c Press, N e w Y o r k (1982). 13. V. D ' A U R O R A , A.M. STERN, and D.S SIGMAN, Biophys. Res. Comm. 80 1 0 2 5 - 1 0 3 2 (1978).
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M. TIEN, L.A. M O R E H O U S E , J.R BUCHER, and S.D. AUST, Arch. B i o c h e m . Biophys. 218 4 5 0 - 4 5 8 (1982). M.F. E M E R S O N and A. H O L T Z E R , J. Phys. Chem. 71 3 3 2 0 - 3 3 3 0 (1967). T.T. H E R S K O V I T S , B. G A D E G B E K U , and H. JAILLET, J. Biol. Chem. 245 2 5 8 8 - 2 5 9 8 (1970). L.S. K A M I N S K Y , R.L. WRIGHT, and A.J. DAVISON, B i o c h e m i s t r y i0 4 5 8 - 4 6 2 (1971). B.M. A N D E R S O N , M.L. R E Y N O L D S , and C.D. A N D E R S O N , Biochim. B i o p h y s . A c t a 113 2 3 5 - 2 4 3 (1966). C-H. C H A N G and J. Yarbro, Life S c i e n c e s 22 1 0 0 7 - 1 0 1 0 (1978).