Mitotic Disrupters from Higher Plants - American Chemical Society

Chapter 18

Mitotic Disrupters from Higher Plants Effects on Plant Cells Kevin C. Vaughn and Martin A. Vaughan Southern Weed Science Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Stoneville, MS 38776

Plant compounds that disrupt mitosis have been well-studied for their effects on animal cells but their effects on plant cells have been relatively poorly described. Most of the mitotic disrupters (e.g. podophyllotoxin, vinblastine, trewiasine) cause effects similar to the well known disrupter, colchicine. Squashes of these treated roots reveal numerous cells in prometaphase. At the electron microscopic level, the chromosomes appear condensed but no microtubules are associated with the chromosomes so that movement to the cell poles is impossible. Nuclear membranes reform around the chromosomes, resulting in oddly shaped, lobed nuclei. Cell elongation is also affected because of disruption to the cortical microtubules that are involved in determining cell shape, resulting in isodiametric cells in the zone of elongation. The combination of cell division and elongation inhibition results in a distinctly swollen club-shaped root. These affects are all due to a direct interaction of the disrupter with microtubules, by preventing further polymerization of tubulin into microtubules. Caffeine appears to affect only telophase by disrupting cell plate formation, probably due to failure of the vesicles to fuse to produce a normal cell plate. Caffeine may also have a direct effect on tubulin, like colchicine. Taxol actually promotes tubulin polymerization into microtubules and stabilization of the microtubules, possibly through interaction with microtubule-associated proteins. Although none of these mitotic disrupters have been utilized in agriculture, a number of these compounds are similar to existing herbicides and, if chemically modified or formulated, may be useful as herbicides. This chapter not subject to U.S. copyright Published 1988 American Chemical Society

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C h e m i c a l s o f p l a n t o r i g i n , s u c h as c o l c h i c i n e , v i n b l a s t i n e / v i n c r i s t i n e , and p o d o p h y l l o t o x i n , h a v e l o n g b e e n u s e d as t o o l s t o study m i t o s i s because of t h e i r a b i l i t y to d i s r u p t t h i s c e l l u l a r process. I t was, i n p a r t , t h i s a n t i - m i t o t i c e f f e c t t h a t p r o d u c e d i n t e n s e r e s e a r c h on t h e e f f e c t o f t h e s e compounds on a n i m a l c e l l m i t o s i s i n p u r s u i t of p o t e n t i a l anti-tumor agents. Surprisingly, t h e s e compounds h a v e b e e n u s e d r e l a t i v e l y l i t t l e w i t h p l a n t s , w i t h the exception of the i n d u c t i o n of p o l y p l o i d y w i t h c o l c h i c i n e . Some o f t h e r e a s o n s f o r t h i s l a c k o f i n t e r e s t may b e due t o t h e r e l a t i v e i n s e n s i t i v i t y o f p l a n t s t o t h e s e compounds ( 1 , 2 ) . G e n e r a l l y 1001,000 X i n c r e a s e o f t h e c o m p o u n d i s r e q u i r e d t o e l i c i t t h e same l e v e l of response i n p l a n t s t h a t ' s obtained w i t h animals. C o l c h i c i n e - i n d u c e d d i s r u p t i o n of m i t o s i s i n p l a n t c e l l s has b e e n t h o r o u g h l y d e s c r i b e d (3) a l t h o u g h t h e e f f e c t s , on p l a n t c e l l s , of o t h e r p l a n t d e r i v e d m i t o t i c d i s r u p t e r s such as v i n b l a s t i n e / v i n c r i s t i n e ( 4 , 5, 6 ) , t a x o l ( 7 , 8, 9 ) , p o d o p h y l l o t o x i n ( 6 ) , a n d c a f f e i n e ( 6 , 1 0 , 1 1 , 12) h a v e n o t b e e n a s e x t e n s i v e l y s t u d i e d . O t h e r p l a n t - d e r i v e d a n t i - t u m o r compounds s u c h as m a y t a n s i n e and t r e w i a s i n e , h a v e b e e n l i t t l e s t u d i e d f o r t h e i r e f f e c t s on p l a n t cells. Many c o m p o u n d s c a n a f f e c t p r o c e s s e s s u c h t h a t t h e c e l l s do n o t e n t e r m i t o s i s b u t r e m a i n i n t h e G (gap) o r S ( s y n t h e s i s ) s t a g e s o f the c e l l c y c l e (13). Most of these e f f e c t s are secondary effects o f t h e s e c o m p o u n d s , t h e r e s u l t o f i n h i b i t i o n o f some o t h e r m e t a b o l i c process r e q u i r e d f o r the entrance i n t o m i t o s i s . This r e p o r t w i l l be l i m i t e d t o t h o s e p l a n t compounds w h i c h d i s r u p t m i t o s i s , i n c l u d i n g c y t o k i n e s i s , by d i r e c t l y o r i n d i r e c t l y i n t e r f e r i n g w i t h stages of m i t o s i s per se. In most c a s e s , t h e s e m i t o t i c d i s r u p t e r s i n t e r f e r e w i t h t h e c e l l u l a r s t r u c t u r e s known as m i c r o t u b u l e s . M i c r o t u b l e s are unb r a n c h e d , h o l l o w , c y l i n d e r s composed o f p r o t e i n s u b u n i t s , t u b u l i n , and p r e s e n t i n a l l e u k a r y o t i c c e l l s ( 1 4 ) . The m i c r o t u b u l e i s a b o u t 25 nm i n o u t e r d i a m e t e r a n d i s c o m p o s e d o f 13 s u b u n i t s w h e n v i e w e d in cross section. I m m u n o f l u o r e s c e n c e m i c r o s c o p y and t r a n s m i s s i o n e l e c t r o n microscopy of p r o t o p l a s t ghosts have r e v e a l e d m i c r o t u b u l e s a s l o n g a s 20 ym i n some p l a n t c e l l s ( 1 5 ) . Microtubules are a s s o c i a t e d w i t h maintenance of s t r u c t u r e t h r o u g h t h e c y t o s k e l e t o n a n d c e l l u l a r movement t h r o u g h t h e s p i n d l e and f l a g e l l a r a p p a r a t u s . The a s s o c i a t i o n o f m i c r o t u b u l e s t o c e l l u l a r s t r u c t u r e a n d movement i s c l o s e l y t i e d t o t h e a b i l i t y o f m i c r o t u b u l e s t o u n d e r g o dynamic a s s e m b l y and d i s a s s e m b l y a t a p p r o p r i a t e t i m e s and p l a c e s i n t h e c e l l . M i c r o t u b u l e assembly occurs by n u c l e a t i o n o f n o n - i d e n t i c a l t u b u l i n s u b u n i t s t h a t e x i s t i n t h e c y t o p l a s m as a f r e e p o o l o f α and £ t u b u l i n , e a c h a t a b o u t 55kD. The t u b u l i n h e t e r o d i m e s f o r m a h e t e r o d i m e r w h i c h a r e a d d e d a t t h e a s s e m b l y end o f t h e " g r o w i n g microtubule. Recent data i n d i c a t e t h a t s m a l l o l i g o m e r s of the t u b u l i n h e t e r o d i m e r s a r e added t o the growing m i c r o t u b u l e end, r a t h e r than i n d i v i d u a l s u b u n i t s ( 1 6 ) . D i s a s s e m b l y o c c u r s p r i m a r i l y a t t h e end o f t h e m i c r o t u b u l e o p p o s i t e t h e a s s e m b l y end. T h i s dynamic p r o c e s s i s r e f e r r e d t o as "treadmilling . A l t h o u g h t h i s i s one o f many m o d e l s t o d e s c r i b e the assembly of m i c r o t u b u l e s , there are other t h e o r i e s that e x p l a i n m i c r o t u b u l e a s s e m b l y as w e l l . Other c e l l u l a r f a c t o r s apparently i n f l u e n c i n g t h e r a t e and e x t e n t o f m i c r o t u b u l e p o l y m e r i z a t i o n and 1 1

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d e p o l y m e r i z a t i o n a r e d i v a l e n t m e t a l c a t i o n s , such as c a l c i u m , and microtubule-associated p r o t e i n s (MAPs). A t p r e s e n t , n o MAPs h a v e been i d e n t i f i e d i n a s s o c i a t i o n w i t h p l a n t microtubules (17) and m i x t u r e s o f o n l y α and 3 t u b u l i n can be assembled i n t o microtubules i n v i t r o ( 1 8 , 1 9 ) . T h u s , t h e r e i s no a p r i o r i r e a s o n t h a t p l a n t c e l l microtubules "must include these non-tubulin proteins to assemble i n t o microtubules. Many f u n c t i o n s i n t h e p l a n t c e l l a r e c o n t r o l l e d b y m i c r o t u b u l e s and these f u n c t i o n s a r e d e f i n e d by t h e v a r i o u s c o n f i g urations of microtubules. These c o n f i g u r a t i o n s a r e most e a s i l y observed i n p l a n t c e l l s by observation o f squashes o f softened c e l l s t h a t have been incubated w i t h a n t i b o d i e s t o t u b u l i n p r o t e i n and t h e n w i t h f l u o r e s c e n t l y t a g g e d s e c o n d a r y a n t i b o d i e s . This allows f o ra three-dimensional display of a l l the microtubules i n the c e l l . C o r t i c a l microtubules occur close t o the c e l l w a l l along t h e p l a s m a membrane ( F i g . 1 A ) . I t i s b e l i e v e d t h a t t h e s e microtubules are involved i n o r i e n t i n g c e l l u l o s e m i c r o f i b r i l s d u r i n g c e l l w a l l s y n t h e s i s a n d i n t h e o r i e n t a t i o n o f new m i c r o f i b r i l s that occurs i n the c e l l w a l l during c e l l elongation (20). S p i n d l e and k i n e t o c h o r e m i c r o t u b u l e s ( F i g . 1C) a r e i n v o l v e d i n t h e movement o f c h r o m o s o m e s d u r i n g m i t o s i s . Phragmoplast microtubules ( F i g . I D ) a r e t h o u g h t t o b e i n v o l v e d i n t h e movement and a r r a n g e m e n t o f G o l g i - d e r i v e d v e s i c l e s a t t h e f o r m i n g c e l l plate. Another m i c r o t u b u l e c o n f i g u r a t i o n , t h e preprophase band o f microtubules ( F i g . I B ) , i s a r i n g of microtubules that appears b e f o r e t h e c e l l i s about t o e n t e r m i t o s i s . The p r e p r o p h a s e b a n d a p p e a r s t o a n d was o r i g i n a l l y t h o u g h t t o p r e d i c t t h e l o c a t i o n o f the a r e a where t h e c e l l p l a t e would form f o l l o w i n g t h e s e p a r a t i o n of daughter n u c l e i a t t e l o p h a s e (21). However, compounds t h a t d i s r u p t l a t t e r s t a g e s o f m i t o s i s c a n a l t e r t h e l o c a t i o n o f t h e new p h r a g m o p l a s t (22) . I t h a s b e e n p r o p o s e d t h a t t h e p r e p r o p h a s e band may s e r v e a s a s t a b i l i z e d t u b u l i n p o o l f o r u s e i n m i c r o t u b u l e ., c o n f i g u r a t i o n s r e q u i r e d i n the subsequent m i t o s i s ( 2 3 ) . 11

A l t h o u g h t h e r e a r e many s i m i l a r i t i e s b e t w e e n p l a n t a n d a n i m a l t u b u l i n t h e r e a r e some s t r i k i n g d i f f e r e n c e s . Electrophoretic s e p a r a t i o n of t u b u l i n i n c e r t a i n g e l systems r e v e a l s that p l a n t t u b u l i n s r u n i n the reverse e l e c t r o p h o r e t i c m o b i l i t y as animal t u b u l i n s ( 1 9 , 24) ( F i g . 2 ) . T h a t i s , t h e o r d e r o f m i g r a t i o n i s a , 3 i n a n i m a l c e l l s a n d $, α i n p l a n t c e l l s . Antibodies to tubulin f r o m many a n i m a l s o u r c e s c a n c r o s s r e a c t w i t h p l a n t t u b u l i n , i n d i c a t i n g t h a t many a n t i g e n i c s i t e s o n t h e t u b u l i n m o l e c u l e s a r e conserved. M o n o c l o n a l a n t i b o d i e s t o b o t h α and 3 t u b u l i n r e c o g n i z e a sequence near t h e carboxy t e r m i n u s o f b o t h p r o t e i n s (25) and t h e s e a n t i b o d i e s r e c o g n i z e t u b u l i n from p r o t i s t s , p l a n t s and animals (e.g. 26, 2 7 ) . Data r e p o r t e d below on t h e s e n s i t i v i t y d i f f e r e n c e s b e t w e e n m i t o t i c d i s r u p t e r e f f e c t s on p l a n t s and a n i m a l s i n d i c a t e t h a t many r e c o g n i t i o n s i t e s f o r t h e s e d i s r u p t e r s m u s t d i f f e r f o r p l a n t and a n i m a l t u b u l i n , however. S p e c i f i c Compounds a n d G r o u p s o f Compounds Colchicine. C o l c h i c i n e ( F i g . 3) i s t h e most w e l l s t u d i e d and w i d e l y u s e d o f t h e p l a n t a l k a l o i d s w h o s e mode o f a c t i o n i s t o disrupt t u b u l i n . This tropolone d e r i v a t i v e i s a three r i n g

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F i g u r e 1. Immunofluorescence microscopy u s i n g a n t i b o d i e s t o t u b u l i n and secondary a n t i b o d y l a b e l l e d w i t h f l u o r e s c e i n on s t a b i l i z e d onion root c e l l s r e v e a l d i f f e r e n t microtubule c o n f i r mations. A. C o r t i c a l m i c r o t u b u l e s a t t h e c e l l p e r i p h e r y . B. A hoop l i k e r i n g o f m i c r o t u b u l e s (arrow) i s c h a r a c t e r i s t i c o f t h e preprophase band. C. A c e l l i n t h e p r o c e s s o f m i t o s i s h a s d i s t i n c t s p i n d l e microtubules that r a d i a t e from the poles of the c e l l and a t t a c h t o t h e chromosomes a t t h e c e l l p l a t e . D. A band of s h o r t phragmoplast m i c r o t u b u l e s appears a t the c e l l p l a t e . The t w o n e w l y f o r m e d n u c l e i (Nu) s t a n d o u t i n n e g a t i v e r e l i e f . A l l X 400.

F i g u r e 2. W e s t e r n b l o t o f c a r r o t r o o t a n d b o v i n e b r a i n e x t r a c t s probed w i t h r a b b i t a n t i s e r a t o sea u r c h i n t u b u l i n i l l u s t r a t e the d i f f e r e n c e i n e l e c t r o p h o r e t i c m o b i l i t y between t u b u l i n from these two s o u r c e s : t h e a , 3 o r d e r i n a n i m a l c e l l s i s r e v e r s e d t o 8, α in plant cells. This a n t i s e r a recognizes the α subunit better t h a n t h e 3. C o n f i r m a t i o n o f t h i s was a c h i e v e d b y i n c u b a t i n g d u p l i c a t e b l o t s i n m o n o c l o n a l anti-α a n d 8 t u b u l i n s .

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podophyllotoxin

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steganacin vinblastine

taxol F i g u r e 3. report.

Structures of the mitotic

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s t r u c t u r e compound i s o l a t e d f r o m b u l b s o f C o l c h i c u m a u t u m n a l e , a u t u m n c r o c u s , a s w e l l a s f r o m o t h e r members o f t h i s f a m i l y and r e l a t e d groups (1). Plants which produce c o l c h i c i n e are extremely r e s i s t a n t t o the a n t i - m i t o t i c e f f e c t s o f t h i s compound, t o l e r a t i n g l e v e l s 100-1,000 t i m e s t h o s e t h a t p r o d u c e m i t o t i c a r r e s t i n s e n s i t i v e p l a n t s p e c i e s (28, 29, 3 0 ) . The m o l e c u l a r m e c h a n i s m f o r t h i s r e s i s t a n c e i s not known. C o l c h i c i n e was f i r s t r e c o g n i z e d b y P e r n i c e i n 1889 a f t e r t h e o b s e r v a t i o n of abnormal m i t o t i c f i g u r e s i n the the i n t e s t i n e of a dog who h a d d i e d a f t e r e a t i n g C o l c h i c u m b u l b s . L e v a n (31) de s c r i b e d the m i t o t i c a b n o r m a l i t i e s c a u s e d by c o l c h i c i n e t r e a t m e n t of A l l i u m r o o t s as c o l c h i c i n e or " c - m i t o s i s " , a n o r m a l m i t o t i c c y c l e up t o p r o m e t a p h a s e f o l l o w e d b y t h e r a n d o m s c a t t e r i n g o f c o n d e n s e d c h r o m a t i d p a i r s ("c-metaphase") ( F i g . 4A). The m i t o s i s p r o g r e s s e s no f u r t h e r , t h o u g h t h e c h r o m a t i d s s e p a r a t e ( " c - a n a p h a s e " ) , and the n u c l e u s r e f o r m s r e s u l t i n g i n a "4c s i n g l e n u c l e u s " ( " c - t e l o p h a s e " ) . The n u m b e r o f c e l l s i n m i t o s i s i n a r o o t m e r i s t e m a p p e a r s t o i n c r e a s e f o l l o w i n g c o l c h i c i n e t r e a t m e n t due t o " a r r e s t " a t p r o m e t a p h a s e and an i n c r e a s e i n t h e t i m e t h a t t h e c e l l i s s t a l l e d at t h i s stage. At t h e e l e c t r o n m i c r o s c o p e l e v e l , t h e chromosomes a p p e a r h i g h l y c o n d e n s e d b u t u n l i k e c o n t r o l c e l l s a t p r o m e t a p h a s e , no m i c r o t u b u l e s a r e a s s o c i a t e d w i t h the chromosomes. A f t e r the f r u s t r a t e d attempt at m i t o s i s , the n u c l e a r envelope reforms around t h e c h r o m o s o m e s , r e s u l t i n g i n a l o b e d n u c l e u s ( F i g . 4B) o r , l e s s f r e q u e n t l y , a m u l t i p l e n u c l e u s i f the chromosomes w e r e w i d e l y separated p r i o r to nuclear reformation. A l l forms of m i c r o t u b u l e s a p p e a r t o b e e f f e c t e d by c o l c h i c i n e t r e a t m e n t . Because of the l o s s o f b o t h s p i n d l e and c o r t i c a l m i c r o t u b u l e s , c e l l s a t t h e r o o t t i p n e i t h e r d i v i d e nor e l o n g a t e . This results i n a conspicuously s w o l l e n (club or spear-shaped) root morphology. C o l c h i c i n e c o m p l e t e l y i n h i b i t s the i n v i t r o assembly of t u b u l i n i n t o m i c r o t u b u l e s f r o m b o t h a n i m a l (32) and p l a n t ( 2 , 33) sources. The q u a n t i t y o f c o l c h i c i n e r e q u i r e d t o i n h i b i t a n i m a l tubulin polymerization i n t o microtubules i s f a r below a 1:1 s t o i c h i o m e t r y b e t w e e n t u b u l i n and c o l c h i c i n e c o n c e n t r a t i o n s . Thus, models t h a t have sought to e x p l a i n the mechanism of a c t i o n of t h i s a l k a l o i d have i n v o l v e d a "capping" concept to e x p l a i n the nons t o i c h i o m e t r i c e f f e c t of c o l c h i c i n e . T h e s e m o d e l s (34) e n v i s i o n an i n t e r a c t i o n o f t u b u l i n d i m e r s o r o l i g o m e r s and c o l c h i c i n e w h e r e , f o l l o w i n g b i n d i n g to the dimer, the c o l c h i c i n e - t u b u l i n complex i s a d d e d t o t h e a s s e m b l y end o f t h e g r o w i n g m i c r o t u b u l e , c a p p i n g t h e m i c r o t u b u l e s o t h a t no f u r t h e r t u b u l i n d i m e r s c o u l d b e a d d e d . As mentioned above, m i c r o t u b u l e s are dynamic s t r u c t u r e s t h a t undergo "treadmilling": a s s e m b l y , o r a d d i t i o n o f t u b u l i n d i m e r s a t one end and d i s a s s e m b l y , o r r e m o v a l o f t u b u l i n d i m e r s f r o m t h e o p p o s i t e end. C o l c h i c i n e , b y b l o c k i n g t h e a s s e m b l y end o f t h e m i c r o t u b u l e , c a u s e s an e v e n t u a l l o s s o f m i c r o t u b u l e d i m e r s t h r o u g h the u n h i n d e r e d d i s a s s e m b l y end ( 3 5 ) . T h e r e i s e v i d e n c e t h a t c o l c h i c i n e t u b u l i n c o m p l e x b i n d s t o t h e d i s a s s e m b l y end a s w e l l a s t h e a s s e m b l y end a s e x c h a n g e o f d i m e r s i s i n d y n a m i c e q u i l i b r i u m a t b o t h a s s e m b l y and d i s a s s e m b l y e n d s ( 3 6 ) . A p h o t o a f f i n i t y a n a l o g o f c o l c h i c i n e (37) has b e e n u s e d t o p r o b e t h e t u b u l i n s u b u n i t (α o r £) t o w h i c h c o l c h i c i n e b i n d s i n i t s

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F i g u r e 4. E f f e c t s o f c o l c h i c h i c i n e (lmM) o n p l a n t c e l l s . A. C h a r a c t e r i s t i c " C - m i t o s i s " r e v e a l i n g " s k i - p a i r s " o f chromosomes (arrows) a f t e r treatment o f onion r o o t s . X 4 0 0 . B. Extensively l o b e d n u c l e u s a f t e r a i n c o m p l e t e m i t o s i s , t h e n u c l e a r membrane r e f o r m s a r o u n d t h e c h r o m o s o m e s t h a t a r e u n a b l e t o move t o t h e poles. B a r = 1 . 0 ym.


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i n h i b i t i o n of microtubule polymerization. O n l y t h e α s u b u n i t was bound by t h i s a n a l o g , i n d i c a t i n g t h a t c o l c h i c i n e b i n d i n g i s p r i m a r i l y associated with this subunit. P l a n t t u b u l i n i s r e l a t i v e l y much l e s s s e n s i t i v e t o c o l c h i c i n e t h a n a n i m a l t u b u l i n (1) and a number o f mechanisms f o r t h i s p o s s i b l e g e n e r a l r e s i s t a n c e have been suggested ( 3 ) . Recent work, u t i l i z i n g p u r i f i e d t u b u l i n from higher p l a n t s , has i n d i c a t e d that p l a n t t u b u l i n h a s a much l o w e r a f f i n i t y f o r c o l c h i c i n e t h a n a n i m a l t u b u l i n ( 2 , 3 3 ) . These data i n d i c a t e t h a t t h e d i f f e r e n c e s between p l a n t and a n i m a l r e s p o n s e s t o c o l c h i c i n e i s due t o t h e t u b u l i n p e r se r a t h e r t h a n u p t a k e , t r a n s l o c a t i o n , and m e t a b o l i s m o f c o l c h i c i n e . O t h e r Compounds t h a t b i n d

1

at the "Colchicine Site ' of

Tubulin

P o d o p h y l l o t o x i n ( F i g . 3 ) , a l i g n a n , i s o l a t e d from t h e r h i z o m e s and r o o t s o f t h e may a p p l e ( P o d o p h y l l u m p e l t a t u m ) h a s a l o n g h i s t o r y a s a f o l k remedy and was s t u d i e d e x t e n s i v e l y a s a n a n t i - t u m o r agent (38). Podophyllotoxin, l i k e colchicine, disrupts mitosis at p r o m e t a p h a s e i n a n i m a l ( 3 9 ) a s w e l l a s i n p l a n t c e l l s ( 5 , 6, 4 0 ) ( F i g . 5A) a l t h o u g h f e w d e t a i l e d d e s c r i p t i o n s a r e a v a i l a b l e . Like c o l c h i c i n e , p o d o p h y l l o t o x i n i s much m o r e e f f e c t i v e i n d i s r u p t i n g m i t o s i s i n animal c e l l s than i n plant c e l l s ( 1 ) . Podophyllotoxin i n h i b i t s c o l c h i c i n e b i n d i n g t o t u b u l i n from a n i m a l s o u r c e s (41) i n d i c a t i n g t h a t t h e b i n d i n g s i t e on t h e t u b u l i n m o l e c u l e i s t h e same f o r c o l c h i c i n e a n d p o d o p h y l l o t o x i n . However, t r o p o l o n e , w h i c h i n h i b i t s c o l c h i c i n e b i n d i n g , does n o t e f f e c t p o d o p h y l l o t o x i n b i n d i n g t o t u b u l i n ( 4 2 ) . These data i n d i c a t e t h a t c o l c h i c i n e a n d p o d o p h y l l o t o x i n h a v e two b i n d i n g s i t e s , one o f w h i c h i s shared (42). C o l c h i c i n e b i n d i n g t o t u b u l i n from p l a n t sources i s u n a f f e c t e d by p o d o p h y l l o t o x i n , i n d i c a t i n g t h a t t h e s h a r e d b i n d i n g s i t e on a n i m a l t u b u l i n i s n o t p r e s e n t o r i s n o t shared i n plant tubulin. S t r u c t u r a l l y , p o d o p h y l l o t o x i n i s q u i t e d i f f e r e n t from c o l c h i c i n e w i t h t h e e x c e p t i o n o f a common t r i m e t h o x y r i n g . This may e x p l a i n b i n d i n g t o t h e same o r s h a r e d s i t e a s c o l c h i c i n e o n a n i m a l t u b u l i n ( 4 3 ) b u t d o e s l i t t l e t o e x p l a i n why i t a p p a r e n t l y d o e s n o t b i n d t o t h e same s i t e o n p l a n t t u b u l i n . T h i s i s e s p e c i a l ly unusual considering the s i m i l a r i n vivo e f f e c t s of p o d o p h y l l o t o x i n i n p l a n t and a n i m a l c e l l s and t h e s i m i l a r i t y o f these e f f e c t s t o that of c o l c h i c i n e (e.g.F i g . 5A). Steganacin ( F i g . 3 ) , a lactone from Stegnotaenia a r a l i a c e a , has a n t i - m i t o t i c p r o p e r t i e s i n animal c e l l s s i m i l a r t o that o f c o l c h i c i n e and h a s b e e n shown t o compete w i t h c o l c h i c i n e f o r t h e same b i n d i n g s i t e o n t h e t u b u l i n m o l c u l e ( 4 4 ) . L i k e podophyllotoxin, steganacin shares the trimethoxy r i n g with c o l c h i c i n e and p r o d u c e s m i t o t i c a r r e s t and i n h i b i t s microtubule p o l y m e r i z a t i o n i n a manner s i m i l a r t o t h a t o f c o l c h i c i n e and podophyllotoxin. No s t u d i e s o f t h e e f f e c t o f t h i s c o m p o u n d h a v e been conducted on p l a n t s t o o u r knowledge. "Vinca

A l k a l o i d s " and

Maytansinoids

The s o - c a l l e d " V i n c a a l k a l o i d s " a r e compounds w i t h p o t e n t a n t i tumor a c t i v i t y and a r e w i d e l y u s e d as c h e m o t h e r a p e u t i c a g e n t s f o r

VAUGHN AND VAUGHAN


F i g u r e 5. A. L i g h t micrograph a r r e s t e d prometaphase a f t e r t r e a t m e n t o f o n i o n r o o t s w i t h 0.1 mM p o d o p h y l l o t o x i n . X 400. Β E l e c t r o n m i c r o g r a p h of an a r r e s t e d prometaphase a f t e r treatment o f C a t h a r a n t h u s r o s e u s w i t h r o o t s 0.1 mM v i n c r i s t i n e . The c h r o m o s o m e s a r e c o n d e n s e d b u t no m i c r o t u b u l e s a r e a s s o c i a t e d w i t h t h e m . m= m i t o c h o n d r i o n . B a r = 1.0 ym. C. A p p a r e n t b i n u c l e a t e c e l l a f t e r t r e a t m e n t w i t h 0.1 mM v i n c r i s t i n e . Note the p a t i a l c e l l w a l l (arrow). Nu= n u c l e u s . B a r = 2.0 ym.


282 1

t h e t r e a t m e n t o f H o d g k i n s d i s e a s e and l e u k e m i a ( 4 5 ) . The m o s t s t u d i e d o f t h e s e a l k a l o i d s a r e v i n b l a s t i n e ( F i g . 3) and v i n c r i s t i n e , both derived from Catharanthus roseus (=Vinca rosea). B e c a u s e o f t h e i m p o r t a n c e o f t h e s e c o m p o u n d s i n c h e m o t h e r a p y , many s t u d i e s have been u n d e r t a k e n t o enhance the s y n t h e s i s , r e c o v e r y , and d e t e c t i o n o f t h e s e c o m p o u n d s , i n c l u d i n g b r e e d i n g p r o g r a m s t o enhance the l e v e l s of these u s e f u l a l k a l o i d s (46). At low c o n c e n t r a t i o n s , b o t h v i n b l a s t i n e and v i n c r i s t i n e i n d u c e t h e same k i n d s o f m i t o t i c e f f e c t s a s c o l c h i c i n e i n b o t h p l a n t s and a n i m a l s ( F i g . 5B) ( 6 , 4 7 , 4 8 ) . Other e f f e c t s of these a l k a l o i d s a r e t h e f o r m a t i o n o f m u l t i p o l a r d i v i s i o n s i n p l a n t c e l l s ( 5 , 49) as w e l l as a m i t o d e p r e s s i v e e f f e c t ( 5 0 ) . In animal c e l l s , high c o n c e n t r a t i o n s o f b o t h o f t h e s e compounds i n d u c e t h e f o r m a t i o n o f p a r a c r y s t a l l i n e a r r a y s o f t u b u l i n b u t t h e s e do n o t o c c u r e v e n a f t e r t r e a t m e n t w i t h 1 mM v i n b l a s t i n e i n p l a n t c e l l s ( 5 , 4 7 ) . Because t h e i n v i v o s e n s i t i v i t y o f p l a n t c e l l s t o v i n b l a s t i n e i s much l o w e r t h a n t h a t of a n i m a l s ( 1 ) , i t has been s u g g e s t e d t h a t p l a n t t u b u l i n m i g h t have o n l y the low a f f i n i t y b i n d i n g s i t e s r a t h e r t h a n the h i g h a f f i n i t y s i t e s of animal t u b u l i n . A l t h o u g h v i n b l a s t i n e can s t a b i l i z e the b i n d i n g of c o l c h i c i n e to t u b u l i n , i t a p p a r e n t l y does not b i n d t o t h e same s i t e s o n t h e t u b u l i n m o l e c u l e ( 5 1 ) . Use o f a p h o t o a f f i n i t y analog of v i n b l a s t i n e reveals that the v i n b l a s t i n e b i n d s t o b o t h α and β t u b u l i n ( 5 2 ) . These d a t a are c o n s i s t e n t with t h e r e p o r t o f two b i n d i n g s i t e s p e r t u b u l i n m o l e c u l e b y L u d u e n a e t a l . (48) u s i n g f l u o r e s c e n c e i n h i b i t i o n as a m e a s u r e o f binding. K r a m e r s and S t e b b i n g s ( 4 ) r e p o r t e d t h a t r o s e u s , the source o f b o t h v i n b l a s t i n e and v i n c r i s t i n e , i s r e s i s t a n t t o v i n b l a s t i n e . I n a r e i n v e s t i g a t i o n o f t h i s o b s e r v a t i o n , we ( 4 ^ ) f o u n d t h a t a l t h o u g h t h i s s p e c i e s i s r e s i s t a n t a t up t o 10 M vinblastine and 10 M v i n c r i s t i n e , h i g h e r c o n c e n t r a t i o n s o f e a c h compound c a u s e d m i t o t i c i r r e g u l a r i t i e s t y p i c a l of that found i n s e n s i t i v e p l a n t s : a r r e s t e d p r o m e t a p h a s e and l o b e d n u c l e i ( F i g . 5 C ) . Neither v i n b l a s t i n e n o r v i n c r i s t i n e a r e h i g h l y s o l u b l e and i t i s l i k e l y t h a t , i n t h e o r i g i n a l r e p o r t o f K r a m e r s and S t e b b i n g s ( 4 ) , t h e l a c k o f e f f e c t a t h i g h e r d r u g c o n c e n t r a t i o n s was n o t n o t e d b e c a u s e t h e d r u g was n o t i n s o l u t i o n . We h a v e f o u n d t h a t d i s s o l v i n g t h e V i n c a a l k a l o i d s i n dimethyl s u l f o x i d e i s necessary to obtain true so l u t i o n s o f t h e s e c o m p o u n d s a t h i g h e r c o n c e n t r a t i o n s and the d i m e t h y l s u l f o x i d e i t s e l f h a d no e f f e c t s a t t h e c o n c e n t r a t i o n s utilized. Another l a r g e group of m i t o t i c d i s r u p t e r s are the maytansinoids which include maytansine ( F i g . 3 ) , i s o l a t e d from M a y t e n e u s , and t r e w i a s i n e ( F i g 3 ) , i s o l a t e d f r o m T r e w i a ( 5 3 ) . T h e s e compounds a r e s t r u c t u r a l l y q u i t e d i s s i m i l a r f r o m t h e V i n c a a l k a l o i d s b u t L u d u e n a e t a l . (48) h a v e shown t h a t t h e y b i n d t o s i t e s t h a t o v e r l a p the b i n d i n g s i t e of the V i n c a a l k a l o i d s . L i k e w i s e , t h e y b i n d i n a m o l a r r a t i o o f two m o l e c u l e s o f m a y t a n s i n o i d : one m o l e c u l e o f t u b u l i n . Despite the overlapping b i n d i n g s i t e s o f t h e V i n c a a l k a l o i d s and t h e m a y t a n s i n o i d s , t h e m a y t a n s i n o i d s do n o t i n d u c e t h e f o r m a t i o n o f p a r a c r y s t a l l i n e a r r a y s o f t u b u l i n n o r do t h e y s t a b i l i z e c o l c h i c i n e b i n d i n g a s do t h e V i n c a a l k a l o i d s (48). U n l i k e many o f t h e o t h e r p l a n t - d e r i v e d m i t o t i c d i s r u p t e r s , the maytansinoids are remarkably e f f e c t i v e at d i s r u p t i n g m i t o s i s i n

18.

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283

p l a n t c e l l s as w e l l as i n a n i m a l c e l l s . Their e f f e c t s a r e v e r y s i m i l a r t o c o l c h i c i n e and o t h e r d i s r u p t e r s o f t h i s c l a s s p r o d u c i n g a r r e s t e d p r o m e t a p h a s e f i g u r e s a n d l o b e d , r e f o r m e d n u c l e i ( F i g . 6). O c c a s i o n a l l y , a few remnant k i n e t o c h o r e m i c r o t u b u l e s a r e n o t e d i n the a r r e s t e d prometaphase f i g u r e s but t h i s i s the o n l y d i s t i n c t i o n b e t w e e n t h e e f f e c t s o f t h e m a y t a n s i n o i d s and t h a t o f c o l c h i c i n e . L a r g e numbers o f m i t o t i c a r r e s t s o c c u r e v e n a g t e r t r e a t m e n t w i t h concentrations o f m a y t a n s i n o i d s a s l o w a s 10 M. Generally 10 -10 M s o l u t i o n s of c o l c h i c i n e are r e q u i r e d f o r s i m i l a r l e v e l s o f m i t o t i c d i s r u p t i o n ( e . g . 4,6). C. r o s e u s . t h e ^ p l a n j : t h a t i s t o l e r a n t t o a l l b u t v e r y h i g h c o n c e n t r a t i o n s (10 -10 M) o f v i n b l a s t i n e a n d v i n c r i s t i n e (47) i s n e v e r t h e l e s s e q u a l l y o r m o r e s e n s i t i v e t o b o t h m a y t a n s i n e and t r e w i a s i n e as o n i o n , a v i n b l a s t i n e - s e n s i t i v e s p e c i e s (Vaughn, V a u g h a n , and L u d u e n a , i n preparation). The d r a m a t i c e f f e c t s o f t h e m a y t a n s i n o i d s a t l o w c o n c e n t r a t i o n s i n d i c a t e t h a t t h e y may b e v a l u a b l e t o o l s t o d i s r u p t the m i t o t i c process w i t h o u t the danger of secondary or n o n - s p e c i f i c e f f e c t s n o t e d w i t h t h e n o r m a l l y h i g h c o n c e n t r a t i o n s o f compounds required to d i s r u p t plant microtubules. Taxol T a x o l ( F i g . 3) i s a d i t e r p e n o i d i s o l a t e d f r o m t h e w e s t e r n yew ( T a x u s b r e v i f o l i a ) a n d o t h e r s p e c i e s o f T a x u s (54, 55) a n d , l i k e t h e o t h e r p l a n t a l k a l o i d s d i s c u s s e d above has shown a n t i - t u m o r a c t i v i t y (54). However, u n l i k e the o t h e r m i t o t i c d i s r u p t e r s , t h i s agent a c t u a l l y promotes p o l y m e r i z a t i o n of t u b u l i n i n t o m i c r o t u b u l e s by l o w e r i n g t h e c r i t i c a l c o n c e n t r a t i o n o f t u b u l i n r e q u i r e d f o r p o l y m e r i z a t i o n r a t h e r t h a n p r o m o t i n g d e p o l y m e r i z a t i o n (56, 57). The a c t u a l m o l e c u l a r m e c h a n i s m f o r t h i s s t a b i l i z i n g e f f e c t i s unknown. The o r i g i n a l l i g h t m i c r o s c o p i c s t u d i e s s u g g e s t e d t h a t t a x o l w o r k s i n t h e same m a n n e r a s c o l c h i c i n e s i n c e t a x o l t r e a t m e n t a l s o produces a r r e s t e d prometaphases. E l e c t r o n and i m m u n o f l u o r e s c e n c e m i c r o s c o p i c s t u d i e s i n d i c a t e d t h a t abundant m i c r o t u b u l e s were found b o t h a t t h e i r n o r m a l s i t e s a t t h e k i n e t o c h o r e as w e l l as a t o t h e r s i t e s i n the c e l l where m i c r o t u b u l e s are not n o r m a l l y found, or a t l e a s t n o t f o u n d i n a b u n d a n c e ( F i g . 7A). I n many c a s e s , t h e microtubules are associated, e i t h e r forming doublets or extensions t h a t l i n k t h e m t o e a c h o t h e r o r t o o t h e r c e l l u l a r s t r u c t u r e s (57). F o i s n e r a n d W i c h e (58) h a v e f o u n d t h a t t a x o l n o t o n l y s t i m u l a t e s the a s s o c i a t i o n of t u b u l i n but a l s o t h a t of the m i c r o t u b u l e a s s o c i a t e d p r o t e i n s (MAPs). I n c u b a t i o n o f m i x t u r e s o f t u b u l i n a n d MAPs i n t h e p r e s e n c e o f t a x o l i n an i n v i t r o m i c r o t u b u l e p o l y m e r i z a t i o n system produced microtubules w i t h extensions that c r o s s - l i n k e d the m i c r o t u b u l e s , s i m i l a r to the extensions observed i n v i v o . Antibody l a b e l l i n g of these microtubules w i t h anti-MAPs i d e n t i f i e d these e x t e n s i o n s a s c o n t a i n i n g MAPs (58). The p r e s e n c e o f MAPs i n h i g h e r p l a n t c e l l s h a s n o t y e t b e e n e s t a b l i s h e d (17), a l t h o u g h c a l m o d u l i n o f t e n forms an a s s o c i a t i o n w i t h p l a n t m i c r o t u b u l e s as d e t e r m i n e d by i m m u n o f l u o r e s c e n c e m i c r o s c o p y (59). Thus, i f t a x o l does cause c r o s s l i n k i n g of m i c r o t u b u l e s b y MAP i n t e r a c t i o n , t h e e f f e c t s o f t a x o l i r i v i v o o n p l a n t c e l l s w o u l d i n d i c a t e t h a t p l a n t s a l s o must h a v e MAPs.

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F i g u r e 6. E l e c t r o n m i c r o g r a p h o f o n i o n r o o t t i p t r e a t e d w i t h l y M trewiasine. Two c e l l s a r r e s t e d p r o m e t a p h a s e (*) a n d a l o b e d reformed nucleus i n which t h r e e p i e c e s a r e seen i n t h i s t h i n section (arrows). B a r = 5.0 ym.

18.

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285

F i g u r e 7. E f f e c t s o f 1 yM t a x o l o n g o o s e g r a s s (A) a n d o n i o n ( B ) . I m m u n o f l u o r e s c e n c e m i c r o g r a p h o f t h e g o o s e g r a s s c o n t a i n i n g two s p i n d l e - l i k e formations, oriented i n d i f f e r e n t d i r e c t i o n s are noted (arrows). In t h i s e l e c t r o n micrograph of onion, m i t o t i s a p p a r e n t l y p r o c e e d s n o r m a l l y as e v i d e n c e d by t h e c e l l i n m i t o s i s and t h e n o r m a l l y f o r m e d n u c l e u s ( N u ) . A=X400; i n Β b a r = 2 . 0 y M .


286

P l a n t m i c r o t u b u l e s have been p a r t i c u l a r l y d i f f i c u l t t o assemble i n vitro. M o r e j o h n and c o l l e a g u e s ( 6 0 , 61) t o o k a d v a n t a g e o f the u n i q u e p r o p e r t i e s and a v a i l a b i l i t y o f t a x o l t o p o l y m e r i z e i t i n vitro. Agents used by o t h e r i n v e s t i g a t o r s t o i s o l a t e and p o l y m e r i z e p l a n t t u b u l i n ( g l y c e r o l o r DMSO a t h i g h m o l a r i t y ) allowed a l i m i t e d polymerization of t u b u l i n i n t o microtubules (60), b u t t h e y i e l d o f m i c r o t u b u l e s i s much l o w e r t h a n w i t h t a x o l . A l t h o u g h no c a r e f u l c o m p a r i s o n o f p l a n t and a n i m a l c e l l s h a s been undertaken, the doses of t a x o l used t o e l i c i t e f f e c t s i n a n i m a l c e l l s ( 5 7 ) a n d i n p l a n t c e l l s ( 7 , 8, 9 ) a r e s i m i l a r , i n dicating similar affinities for tubulin. I n t e r e s t i n g l y , not a l l p l a n t s a r e a f f e c t e d t o t h e same d e g r e e b y t a x o l t r e a t m e n t . Onion, a s p e c i e s o n w h i c h many m i c r o t u b u l e d i s r u p t e r s t u d i e s h a v e b e e n p r e f o r m e d , shows v i r t u a l l y no change i n m i c r o t u b u l e o r i e n t a t i o n o r o t h e r m i t o t i c i r r e g u l a r i t i e s e v e n a f t e r 24 h o f t r e a t m e n t i n 10 yM t a x o l ( F i g . 7B) e v e n t h o u g h o t h e r s p e c i e s show l a r g e i n c r e a s e s i n the m i t o t i c i n d e x and a b n o r m a l l y o r i e n t e d m i t o t i c f i g u r e s . There are s e v e r a l p o s s i b i l i t i e s that e x p l a i n these data. Substances such a s t a x o l , p r e s e n t i n l o w c o n c e n t r a t i o n s i n t h e c e l l , may b e u s e d b y the c e l l t o s t a b i l i z e the m i c r o t u b u l e s during t h e v a r i o u s conformations that are required to maintain a proper c e l l cycle. Onion may h a v e i t s t u b u l i n s t a b i l i z e d b y a c e l l u l a r a g e n t s i m i l a r t o t a x o l , r e n d e r i n g i t l e s s s e n s i t i v e t o an e x t e r n a l s t a b i l i z e r . The p r e s e n c e o f a s t a b i l i z e r may e x p l a i n t h e r e l a t i v e i n s e n s i t i v i t y o f o n i o n t o a number o f m i t o t i c d i s r u p t e r h e r b i c i d e s , s u c h a s dimethyltetrachloroterephthalate (DCPA) ( 6 7 ) . Caffeine

and R e l a t e d

Methylxanthines

C a f f e i n e ( F i g . 3) i s a n o x y p u r i n e c o n t a i n e d i n f r u i t o f t h e c o f f e e plant (Coffea arabica). Unlike the other a l k a l o i d s which disrupt m i t o s i s , t h e m e t h y l x a n t h i n e s , o f w h i c h c a f f e i n e i s a member, a f f e c t the c o m p l e t i o n o f c y t o k i n e s i s a t t e l o p h a s e and n o t t h e f o r m a t i o n the m i t o t i c s p i n d l e a t prometaphase. The p r e c i s e m e c h a n i s m b y w h i c h c a f f e i n e p r o d u c e s t h i s e f f e c t i s n o t k n o w n . T h e r e i s some e v i d e n c e , a l t h o u g h r e l a t i v e l y s m a l l when compared t o t h e o t h e r m i t o t i c i n h i b i t o r s , t h a t c a f f e i n e a c t s d i r e c t l y on t u b u l i n . Other e v i d e n c e i m p l i c a t e s an e f f e c t o f c a f f e i n e on c a l c i u m concentrations and v e s i c l e f u s i o n a t t h e c e l l p l a t e . As more i n f o r m a t i o n i s c o m p i l e d on t h e m o l e c u l a r p r o c e s s e s i n v o l v e d i n c e l l p l a t e f o r m a t i o n , t h e m o l e c u l a r s i t e o f a c t i o n o f c a f f e i n e w i l l no d o u b t be e l u c i d a t e d as w e l l . Treatment o f root meristem c e l l s w i t h c a f f e i n e r e s u l t s i n normal m i t o s i s but apparent f a i l u r e of c y t o k i n e s i s produces large numbers o f b i n u c l e a t e c e l l s and c e l l s w i t h i n c o m p l e t e c e l l p l a t e s ( 6 , 10, 12, 6 3 ) . V e s i c l e s d e r i v e d f r o m t h e G o l g i a p p a r a t u s a p p a r e n t l y move t o w a r d t h e c e l l p l a t e b u t t h e v e s i c l e s d o n o t f u s e s o t h a t an i n c o m p l e t e , r e t i c u l a t e c e l l p l a t e r e s u l t s ( F i g . 6 ) . I n e x t r e m e c a s e s , no c e l l p l a t e i s formed and b i n u c l e a t e c e l l s r e s u l t (Fig. 8 ) . C a f f e i n e i s a p o t e n t i n h i b i t o r o f cAMP p h o s p h o d i e s t e r a s e i n a n i m a l c e l l s ( 6 4 ) and i t was assumed t h a t t h e c a f f e i n e e f f e c t on p l a n t c t y o k i n e s i s w a s d u e t o e l e v a t e d l e v e l s o f cAMP a n d d e c r e a s e d l e v e l s o f c a l c i u m a f f e c t i n g t h e f o r m a n d f u n c t i o n o f membrane

18.

VAUGHN AND VAUGHAN


F i g u r e 8. A f t e r 0.1 mM c a f f e i n e t r e a t m e n t t h e c e l l s a r e binucleate. V e s i c l e s ( a r r o w s ) o f abnormal s i z e and c o n t e n t a r e f o u n d s e p a r a t i n g t h e two n u c l e i ( N u ) . Bar=5.0ym.

287

288


v e s i c l e s at the c e l l p l a t e (10). However, the overwhelming e v i d e n c e i n d i c a t e s t h a t cAMF d o e s n o t e x i s t i n h i g h e r p l a n t s ( 6 5 ) . A r o l e f o r c a l c i u m ( 1 0 , 1 1 , 12) and p o s s i b l y m a g n e s i u m ( 1 1 ) h a s b e e n d e m o n s t r a t e d f o r c y t o k i n e s i s i n p l a n t c e l l s s u g g e s t i n g an e f f e c t o f c a f f e i n e on c a l c i u m l e v e l s a t t h e c e l l p l a t e . Other m i t o t i c d i s r u p t e r s can cause c a l c i u m i n b a l a n c e ( 6 6 ) . H o w e v e r , i t i s now b e l i e v e d t h a t , a l t h o u g h the e f f e c t s are r e a l , they are not the primary cause of m i t o t i c d i s r u p t i o n . E f f e c t s of m i t o t i c d i s r u p t e r s are g e n e r a l l y determined i n f i x e d s p e c i m e n s and t h e s e q u e n c e o f e v e n t s a r e o f t e n i m p l i e d b a s e d upon s t a t i c e l e c t r o n m i c r o g r a p h s . B o n s i g n o r e and H e p l e r (12) observed l i v i n g , d i v i d i n g stamen h a i r s of T r a d e s c a n t i a t h r o u g h m i t o s i s a f t e r t r e a t m e n t w i t h c a f f e i n e t h r o u g h the use of N o m a r s k i d i f f e r e n t i a l interference microscopy. Quite s u s p r i s i n g l y , they o b s e r v e d t h a t a n a p p a r e n t l y n o r m a l c e l l p l a t e was f o r m e d b u t that t h e p l a t e was s u b s e q u e n t l y d i s t u r b e d . T h i s may be due t o t h e f a i l u r e o f t h e v e s i c l e s t o f u s e p r o p e r l y so t h a t a s t r u c t u r e that a p p e a r s i n t h e m i c r o s c o p e a s a w a l l i s l a i d down and l a t e r d i s p e r s e s as the p h r a g m o p l a s t m i c r o t u b u l e s d i s a p p e a r . Because phragmoplast m i c r o t u b u l e s d i s a p p e a r from the c e n t e r of the c e l l f i r s t , the images of p a r t i a l c e l l p l a t e s p r e s e n t o n l y a t the edge o f t h e c e l l ( 1 0 ) may be explained. A d i r e c t i n t e r a c t i o n b e t w e e n t u b u l i n and c a f f e i n e and related a l k a l o i d s has a l s o b e e n n o t e d i n a number o f s t u d i e s . In the green a l g a Chlamydomonas p r e s u m p t i v e t u b u l i n m u t a n t s t h a t a r e r e s i s t a n t to c o l c h i c i n e e x h i b i t heightened s e n s i t i v i t y to c a f f e i n e (67). Chlamydomonas f l a g e l l a a r e a c o n v e n i e n t e x p e r i m e n t a l s y s t e m f o r s t u d y i n g e f f e c t s o f compounds on m i c r o t u b u l e s . The caffeine a n a l o g , i s o b u t y l m e t h y l x a n t h i n e (IBMX), caused f l a g e l l a r resorption, and, l i k e o t h e r m i c r o t u b u l e d i s r u p t e r s ( 6 8 ) , caused changes i n the mRNA l e v e l s f o r f l a g e l l a r p r o t e i n s s u c h a s t u b u l i n ( 6 9 ) . Moreover, M o r e j o h n ( u n p u b l i s h e d ) h a s f o u n d t h a t IBMX i n h i b i t s t h e i n v i t r o polymerization of t u b u l i n i n t o m i c r o t u b u l e s , although the l e v e l s r e q u i r e d were h i g h e r than that f o r o t h e r m i t o t i c d i s r u p t e r s . S u p p o r t i n g these i n v i t r o data are the i n v i v o s t u d i e s of J u n i p e r and L a w t o n ( 7 0 ) . These workers observed that c o r t i c a l m i c r o t u b u l e s of p r i m a r y c e l l w a l l were r e l a t i v e l y c a f f e i n e - i n s e n s i t i v e but c o l d - s e n s i t i v e whereas the c o r t i c a l m i c r o t u b u l e s of the secondary c e l l w a l l were c a f f e i n e - s e n s i t i v e but c o l d - i n s e n s i t i v e . These d a t a w e r e t h e f i r s t t o i n d i c a t e t h a t t h e r e may b e d i f f e r e n c e s b e t w e e n m i c r o t u b u l e s of d i f f e r e n t c e l l types, at l e a s t i n t h e i r s e n s i t i v i t y to d i s r u p t i o n . V a u g h n £t a l . ( 6 ) o b s e r v e d t h a t the d i n i t r o a n i l i n e - r e s i s t a n t b i o t y p e o f E l e u s i n e i n d i c a ( w i t h an a l t e r e d t u b u l i n c o m p o s i t i o n ) was much m o r e s e n s i t i v e t o c a f f e i n e and r e l a t e d a l k a l o i d s t h a n t h e s u s c e p t i b l e b i o t y p e b u t , b e c a u s e t h e r e s i s t a n t b i o t y p e made a b n o r m a l c e l l w a l l s , i t c o u l d n o t be det e r m i n e d w h e t h e r t h i s was a d i f f e r e n t i a l c a f f e i n e e f f e c t o n t u b u l i n o r an e x a c e r b a t i o n o f an a l r e a d y a b n o r m a l s i t u a t i o n .

Comparison of H e r b i c i d e s w i t h

Plant

Derived M i t o t i c

Disrupters

I n g e n e r a l , p l a n t - d e r i v e d m i c r o t u b u l e d i s r u p t e r s a r e much more e f f e c t i v e on a n i m a l t h a n on p l a n t c e l l s ( 1 ) , w i t h t h e e x c e p t i o n

of

18.

VAUGHN AND VAUGHAN


289

t h e m a y t a n s i n o i d s and t a x o l as d e s c r i b e d a b o v e . What a r e t h e c h a r a c t e r i s t i c s t h a t mark an e f f e c t i v e m i t o t i c d i s r u p t e r f o r p l a n t c e l l s and t h o s e t h a t a r e e f f e c t i v e f o r a n i m a l c e l l s ? Usually e f f e c t i v e animal c e l l m i t o t i c d i s r u p t e r s are those which have at l e a s t a f a i r s o l u b i l i t y i n w a t e r and a r e o b t a i n e d f r o m p l a n t s ( o r s h a r e a s i t e o f a c t i o n w i t h a n a t u r a l p l a n t compound). At the other extreme, e f f e c t i v e p l a n t c e l l m i t o t i c d i s r u p t e r s g e n e r a l l y h a v e a low w a t e r s o l u b i l i t y and a r e c h e m i c a l l y - d e r i v e d ( T a b l e 1 ) . Compounds t h a t a f f e c t b o t h p l a n t s a n d a n i m a l s , i n c l u d i n g t h e c o m p o u n d s p r o p h a m and c h l o r p r o p h a m , a r e f o r m u l a t e d f o r b o t h h e r b i c i d a l a n d i n s e c t i c i d a l u s e , and a r e g e n e r a l l y i n t e r m e d i a t e i n water s o l u b i l i t y . P a r t o f t h i s d i v i s i o n may r e f l e c t t h e n e e d s f o r t h e s e compounds r a t h e r t h a n s p e c i f i c m i c r o t u b u l e s i t e s t h a t a r e a t t a c k e d by each d i s r u p t e r . For example, the d i n i t r o a n i l i n e h e r b i c i d e s are h i g h l y i n s o l u b l e compounds t h a t a r e i n c o r p o r a t e d i n t o t h e s o i l p r i o r to crop p l a n t i n g . Crop p l a n t s t h a t are t o l e r a n t of these h e r b i c i d e s are g e n e r a l l y l a r g e seeded, l i p i d - r i c h p l a n t s t h a t can s e q u e s t e r t h e h e r b i c i d e i n l i p i d b o d i e s ( 7 1 ) away f r o m t h e s i t e o f a c t i o n , the microtubule. S e n s i t i v e weed s p e c i e s , c h i e f l y t h e g r a s s e s , a r e s m a l l s e e d e d a n d h a v e much l o w e r l e v e l s o f l i p i d and are unable to sequester the h e r b i c i d e . The s o l u b i l i t y c h a r a c t e r i s t i c s o f t h i s g r o u p o f h e r b i c i d e s r e n d e r s some s e l e c t i v i t y , a l l o w i n g them t o be u s e f u l h e r b i c i d e s . Thus, the s e l e c t i v e h e r b i c i d a l p r o p e r t i e s o f a c o m p o u n d may be t h e r e a s o n f o r t h e l o w s o l u b i l i t y o f many o f t h e p l a n t - s p e c i f i c d i s r u p t e r s , r a t h e r t h a n c h e m i c a l d i f f e r e n c e s r e l a t e d to plant-animal d i f f e r e n c e s i n t h e i r t u b u l i n . P o t e n t i a l Use ture

of Plant-Derived

1

Microtubule

Disrupters

in Agricul-

T h i s symposium s purpose i s to h i g h l i g h t the p o t e n t i a l of n a t u r a l p r o d u c t s as a g r i c u l t u r a l a g e n t s . At p r e s e n t , none o f the p l a n t d e r i v e d m i t o t i c d i s r u p t e r s are used to s o l v e crop problems but t h e r e i s a p o t e n t i a l f o r some u s e s . T a x o l has b e e n shown t o p r o t e c t a n i m a l c e l l s f r o m s u b s e q u e n t t r e a t m e n t w i t h m i t o t i c d i s r u p t e r s s u c h as c o l c h i c i n e and m a y t a n s i n e (34). B e c a u s e a n u m b e r o f h e r b i c i d e s a r e m i t o t i c d i s r u p t e r s , we (72) i n v e s t i g a t e d w h e t h e r t a x o l c o u l d p r o t e c t s u s c e p t i b l e E l e u s i n e i n d i c a (goosegrass) from subsequent treatment w i t h the d i n i t r o a l i l i n e h e r b i c i d e s t r i f l u r a l i n or o r y z a l i n . Treatment of t h e s u s c e p t i b l e b i o t y p e w i t h s u b l e t h a l l e v e l s o f t a x o l f o r 24 h a n d s u b s e q u e n t t r e a t m e n t w i t h 10 yM t r i f l u r a l i n o r 1 yM o r y a l i n r e s u l t e d i n no a b n o r m a l m i t o s e s . G r a s s e s are v e r y s e n s i t i v e t o most m i t o t i c d i s r u p t e r h e r b i c i d e s and t r e a t m e n t o f g r a s s s e e d l i n g s w i t h a p r o t e c t a n t s u c h as t a x o l w o u l d a l l o w s e l e c t i v e weed c o n t r o l o f weed g r a s s e s i n c r o p g r a s s e s , s u c h as J o h n s o n g r a s s (Sorghum h a l a p e n s e ) c o n t r o l i n sorghum (Sorghum b i c o l o r ) , an e s p e c i a l l y d i f f i c u l t h e r b i c i d e problem. An e f f e c t i v e m e t h o d o f i m p l e m e n t a t i o n might i n v o l v e simply t r e a t i n g dry seeds w i t h a s o l u t i o n of t a x o l or a t a x o l d e r i v a t i v e and t h e n p l a n t i n g t h e t r e a t e d s e e d i n t o s o i l incorporated with d i n i t r o a n i l i n e herbicides. Of c o u r s e , t h e s c a r c i t y o f t a x o l a t p r e s e n t makes s u c h a p r o p o s a l seem q u i t e ridiculous. The d e v e l o p m e n t o f a n i n e x p e n s i v e s y n t h e s i s o f a

III

on plants

Amiprophosmethyl

Pronamide

Dinitroanilines

E f f e c t i v e only

propham

chlorpropham

maytansinoids

taxol

I I E f f e c t i v e both on p l a n t s

oncodazole

griseofulvin

podophyllotoxin

vinblastine

colchicine

and a n i m a l s

I Much more e f f e c t i v e on a n i m a l s

Group

from b o t h c h e m i c a l and p l a n t

than

added;

water

^ 2ppm w i t h o u t o r g a n i c s o l v e n t s none a r e p l a n t - d e r i v e d

M a i n l y l i p i d s o l u b l e ; maximum solubility usually

sources; s o l u b i l i t i e s generally less group I b u t g r e a t e r t h a n group I I I

Obtained

Mainly plant-derived, at least moderately water soluble

Characteristics

T a b l e 1. G r o u p i n g o f m i t o t i c d i s r u p t e r s b a s e d u p o n d i f f e r e n t i a l s e n s i t i v i t i e s o f p l a n t s and a n i m a l m i c r o t u b u l e s t o t h e s e compounds

18. VAUGHN AND VAUGHAN

Mitotic Disrupters from Higher Plants 291

compound with the properties of taxol is something for synthetic organic chemists to consider, however. Another potential approach involves chemical modification of the disrupter by adding other substituent groups that would render them less toxic to humans, and increase the chance for selectivity to and within groups of plants. An example will make this more clear. Paraquat is a non-selective herbicide that acts by accepting electrons from the primary acceptor of photosystem I. A parquât radical dication results which reacts with molecular oxygen to generate superoxide and other toxic oxygen species such as hydroxyl radical and peroxide (73). Some years ago ICI introduced a bis-carbamoylmethyl derivative of paraquat called morfamquat. Unlike paraquat, this compound was very effective in controlling broad-leaved weeds but was very ineffective in controlling monocots. Although the exact mechanism for this discrimination is not known (74), the most believable explanation for this discrimination is that dicots are able to hydrolyze the carbomyl group from morfamquat, yielding a molecule that is much easier to move across membranes in the cell. Further decarboxylation would give rise to the parent compound paraquat. By using similar modified forms of plant derived mitotic disrupters, it may be possible to render these disrupters non-toxic to man, increase their efficiency at entry into the plant by increasing their lipophilicity, and perhaps achieve the selectivity desired of a herbicide. Acknowledgment s Thanks are extended to R. H. Jones and P. Wolfe for technical assistance. The NIH kindly provided taxol for our studies. R. Powell and R. Luduena provided generous samples of trewiasine and maytansine, respectively. This work was supported in part by a USDA Competitive Grant (86-CRCR-1-1933) to K. C. V. Literature Cited 1.

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