Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
A_~.~ A ~ L ~ ~/ ' L .~r 1-1 ~ I VL~ I ~AnV1~ 1nh~ U ~
2069122
~I~DS ~1) CO~O~ oN8 P'~ P~bU~N oF ~AtlOI"
TAXOL ~R~CCUR~R8 AND ~ IVAT~ O~
~ROM Y~NT TI 8
~O~ 'rP.~ SN' ~
~h~ inv0n~ion deAl~ Wlth th~ p~odu~ n ~na uae t~r
c~nd a6~ u~pen~ion culture~ b~ ~IXU~ gro~n on
~3mbran~ , ln 6haker a~llt~ a, ~nd ln ~ior~aatoXq ~or
th~ l?rodu~ion ~t t~xo~, ~r~o~s~r~ ~n~ lnt~ iAt~
o t)l~ bloo~s~ioal pathway r~l~ultlng ln t~ol ~ro~uc~lon, ~n~
~a~lv~ d~rlvAtiv~ o~ taxol, includln~ o~p~alo~nnino an~
~c~atin. T~xol and ~axol-x~l~.t~d ~o~our~ haw ~c~ait:lv~
~t~vlty ln aana~r mod~ y~ ~nd ~l~rotu~
~t~112ing aa~Ay~, ~h~ pound~ pot~
power~ul ah~mot~erapeutla ag~nt~.
I~xol tN~C-125g73) la a oyta~oxio di'cQ~p~n~ wlsh a
oom~ x ch~mis~ry, initi~lly i~ol~ta~ froPI th~ ~t~ rX
o~ t~a woat~rn y~, q'~U3 brovl~olls Nut~. ~ by t~Ani ~nd
}o Go-wortcerB ~n ~g71. ~ub~guen~ly, ~t nas be~n ~2~own~co
~ pr~nt in t~o ~ a~ ~ll a~ 'ch~ roo~ o~ tb~
p}~nt ~Wltl7~ru~ 90; ~7ld~aok, 1~90~. It i~ the
~nly pl~nt ~ro~ur~t ~cn3wn to ~r~m~t~ a~ly t~
microtubul~ and inhl~lt ~ho tubulln d~u~ ly ~roo~u~,
~nd a~aa~s to ~Q th~ prototy~o o~ ~ new ola~ o~ ~na~r
ah~mothera~ tlc ag~n~ u~n~a~ an~ Cord~ll, 19~5~
~n addltion to t~xol "nany othOr clo~qly r~lat~d
cyto'coX$c taxan~ dQrivat~vB~ h~ s~n ~aol~ted rGm th~
Uonu~ Nc~auqhlln ~t ~l., 1981; ~ t al., lg8~J
5~in~oton ~t ~ lq92; ~IUAng ~ al . ~ 1 9B6) ~ ~ut ~r
rotubul~ ~t~b~ g capn~ ky ~n~ ch~eth~ra~tic
4~ ar~ not xnoun.
T~xcl ~xhl~lt~ aot~lrlty ~ln~ o K~ ~n~r cell
l~no u~e~ to scra~n ~o~ ~ot~n~ial ch~m~r~pou'cia ~b~erlto
ln till!ll~4 ou~ture. Durlng ~x~alinlea~ ~v~lu~t~on ~tu~
lt h~ boon ~hown to h~tQ a~lvity ~galn~t b~'ch ~ n~
~lld t~mor lin~s un~ oQll lln~ ~W~n~
19?1; suffnQ~ And cord~ll, 19~$J D~n~how~r ot al., 19~7~,
P~aentl~ t~xol ha~ b~/an ~hown ~o ~ act~v~ ln t~e o
3 o human Ovari~J~ ca~cinom~ x¢n~ t mod~l and ~:~ hav~
.
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~ ~Y~: I ~Y~ U 2 1 . ~ . .1 . J _ I ~ ,, ~,, ~ ~, ", , J I I t ~ 't '' -t ' t C~
- 2 - 20~9122
~roml~e ln ~h~ troa~Qnt o~ a~A~c6~ ragY~iV~, an~
drug-r~actory ~vArlan d~ aeuiro ~ ., lgi~g;
Thl~n, ~t ~1., 1990~ .
~xol 1~ ~ potent lnh~b~t~ btr ~Q~ 11cat~on th~t
act~ aa an ~n~1mltot1~ ~gent, blockln~ ~ol1~ ~n th~ oa~M
pha~ o~ tha o~l~ (SCbl~ ~nd t~rowit~, 19~0J ~orowlt4: ot
~l., 1986~. ~t 1~ uni~a ln it~ ab~ y to b~n~
gtoiahi~mQ~r~ y to ~olymeriz~d ~bulin ~oth in ~rltro
~T~d ~3~ VlYo, an~ lndua~- ths a~bly o~ ~l~lln ~0 orYD
lolum-~table ~ç~grog~ as~o~cu~ul~ ~ruatur~ h~
~tru~ur~ rl~si~t ~ol~erlz~tlon L~y d~lutlon, ~alc~um
ion~, cold ~nd a s~u~bor o~ m~ero~ulo-d1cru~tln~ ~ruç~
M~crotubul~s a~am~l~ tubulln ~n th~ ~b~sfloQ ot sxo~no~l~
~uano~ine 5 ~ phs~h~ or ~ crotubul~-a~ooiat~d
protl~lnB A~ lo~t t8mper2tur~ ch~P~ ~nd 130rowitz, 1Ç~O~
~nrn4~ nd ~rowlts, 198~ ~ow~ nd q'l~dR~ , 198~)
and ~3B~ ~I alln~ o~l ~tudiso l~ 3n
conduoted wlth ~aXC~. llo~v~r, ~ 0~ ~upply o~
taxol h~- pr~l~lud~d ~xt~e~ llnlq~l ~'CUdl~. 13acau~
~0 there is a ~tron~ po~ ility th~t t~#:C~l w~ ho~
8isni~1aant aCt~Vity ~ln~t ~ d~ r~ng~ o~ aan~ar~, lt
~ ~ cr~a ~ al to snaka ~ bl~ t~ aup~ nt
t~u~ ~ultur~ t~ohnlguo~ haY~ thu ~t~n~i~l to pr~dua~
an abund~nt su~}~ly o~ nd ~ iolog~ ~ally ~ctive
2 5 ~o~pound~ and ~ ur~or~ . In ~d~lltlon t~ ~rovi~ing a
r~p~dly avai~ R and ~r~n~abl~ ~oura~ t~xol, ~not~r
b~n~lt a~ t~6~ue cultura a~ urcD i~ tho ~n~ ti~r~
o~ y~w tre~, a ~rg~ r oP whi~h r.eed ~o b~ h~rvn~4fl
~n or~er to l~o~at~ the kllo~ t~ ti~ pr*~nt~y
n~dod ~or cl~n~e~l u~.
~xol haJ ~n rou~in~ly ~r~ct~ ~ro~ ~ch~ drl~d ~t~D~
b~ o~ T. b~-v~ n ~ tlon ~XQ~ al~o ~
i~olatod rrcm ~ov~r~l othor ~pac~o~ ~f y~ ~g~nU~ T~uo,
~a~lly ~2xaceae), ~r~alu~ing ~. b~cGzl~a and ~ p1dati}
(Southton ~md Bucxlnqh~ lg8~1 and hlor~ r~cently ~
c~aaens~a c~r. Capitata Qnd ~. m0d~ cJY~ naltor~ Lnd
k~ll twit:l~eru3? 1~ A~ 90) . 5q~ d~
"~
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2V69122
h?~v~ n ~hown t~ h~v~ hlgh~r ~e~ o~ t~xol tl~n r~xuR
bre~-~lfsY~a ~blQ ~).
Although t~xol h~ n ~xtraa'co~l t~ tu~al
~oura~, ex~r~t~ on ha~ n ~ ~ely p~ ta~lng, And
pr~duo~ ~ro~ly lnc~decluat~ or b4t~ pro~ and
ant~ t~v~ naod~ U~8 ~h~ae ~.mpo~nd~ ~r~ ~o~
~x~ ely low cona~nt~ n~ in ~lant~ tO.Ol~ dry welg~t
ba~lo) . Although tn~ ~a~onal C~ncor $n~t~t~ h~ ba~n
~b~ to ~?~c~C oVor g~,004 ~o~nd~ d ~. b~c~oll~
ba~k, only l-a k~ o~ t~ol h~Y~ ba~r ~xtra~t~d durlng
tha ~a~ ~lv~ y~ar~ ~u~n~ 9D) . ~a6ed on an ~Y~
at~d yield of O . Oâ9~ col on ~ ~!lry ~ig~t ~oa~ it
1~ 0~1m~ted thA~ ~ to~l o~ 720,000 pouna4 ~ bark or
3~, 000 tr~ woul~l n~od ~ b4 h~rv~t~ ~0 ob~aln
~u~ol~nt ~xol ~or ~ dlath ph~r~ utl~al UB~D, if~ tho
nati~ 'r. ~rD~l~oll~ i~ t~ lo c~ur~ n~d~r,
1~0) .
in g~n~rnl hav~ ~ory ~14w ~rowth rAtoq ~n~ lony
prop~tlon ~rio~l~;. Th~ lt dl~flc~lt t~ i~l~0nt
br~adinq S;rogram~ aim3d at ~cra~nlns~ ~r ~p~ale~ pr~duoin~
;3n 2~bundant ~upply o~ thcoo ao~po~ u~ to ~h~ V~y
~low ~o~rth o~ 1l11 y~w~ d the lo~r y191d~ 0~ e~X~l
obt~in~d, th~r~ la wid~r~ad ~on~a~n th4t a~t~nat~
~ut~ ~rQ n~d~d to ntainta~n an aS~ t~ and c~ntl~u~u~
~u~ply o~` thi~ d~ug. ~he~i~t~ al~ O~ t` the w~rl~ h~
bo-n lnvolv~d in ~n ~tt~mpt eO whl~ ot~ o~
t~xol t~u~rl~tn-vo~g~le~ ~9t ~ 8t), ~ut tha~s ~oxt~
hav~ onl~ ~e~ ~lith llm;t~ . EvQn ~ ~o~o o~ th~
V~rioUE~ ~trat~ empl~y~d p~u~e ~exol I thoy naqre
llttl~ pr~ a~l v~lu~ oau~0 ~h~y ara ~lilc~y to
~urnlP~ t~xol ~nd t~xo~ t~ co~po~ ln ~or~ ~hsn
aa flmoun~cs (D~ni~ ~t al., 1988~ .
Con~1ictf~ BXl~t ~tw~on th~ nb~ ~or l~rq~ un~
o~ t~xol, ~n ~Ytrar~ly ~ hamoth~r~p~utla ~n~
3~ an~ tho cona~rn o~ ~nviyon~Qnt~ t~ to pr~a~r~e th~ y~w
tr~s (g~rAU~ T~xu~ a~lly ~!~Yaa~a~3 whlch at ~r~e-t Ar~
t~lO only 60urc~ o~ t~x~ r~ov~r, A ll~r~OU4 llmltat~on
ln t~xt~A~tlng t~xol ~lr~c~ly ~ral~l y~w tree~ th~ thl~
. ~
,. :.,
.
2~122
hod ha~ only vexy low yl~la, ir!~hf~ u~lc~ to ~at~ ~y
n~od~ of ~n~or ra~aroh~r~ hQ~a con~ t~r~g allni~l
tri~l~ on ~h~ a o~ ~xol. ~v~n nl~ro ~v~x~ ~ho~t~g~
o~ taxo~ would on~ p~ re~t~nt or oAn~s
wi~h t~ol wA~; init~t~t~a, Bu~h ~hc~rtR~ wo~
corr6~1~ta~ wlth ~roh~ itl~f#ly hlç~h oo~t~ o~ the flru~.
q~hRrla i8 ~n lm~d~ ate e~ aruclal n~od to ~wlo~
rapidly r~prod~aiblo, contlnuou~ly ~vn~ lAbls~, r~ w~Sl4
~ouros~ o~ t~s~nl ~nd t~ol-xoL~t~ c~ und~D, ~ h~ or
rg~ ~a~ produatl~n. ~saaU~R ~bW ~o g~a~ y
~lowly, plnntlng ~or~ ysw tr~ not ~ ~rb~i~ing
~ol~tion. Ch~mic~ h~ a~ no~ b~n ~oc~QB~ul.
T~x~nc rlng-~ontalnlng ~lX~lo~d co~aundl~ hav~ b00n
obta~n~d trom coll ~ulturR~ o~ T. ~v~ol~, bu~ ~on~-
term, ~ablR gro~h ~ ~uch Cu~tur~, or o~ ~Bt~ d
a~ll llne~ of ;~rly ~ hav~ not ~n r~ rt~ ~Chr~tQn
~t ~ 19~1) .
~h~ mathod~- an~ s:ompo~ltlbn~ o~ pr~n~ i~v-Dn~ n
ar~ ~ no~utlon ~o th~ psobl~ of tax~ up~ly b~a~u~ y
ra~& to ~ UC~ cult~sl o~ ~AXU~ tt~ ~v~l~p Htabl~ lon~-
t~ ~æll t ln~ wt~lc~ prc~duc~, ~v~n 8ve~prndl~o~, tAxol,
l~no~ to u~a t~i~ tho~ coll lin~ r~ o~ to ~?rodu~
tA~ nd ta~col-ralat~d a~m~oUn~; in la~g~ t~ o
th~n ar~ ~08~ With curxon~ ~axol-5~rodu6;tl0n mb~
~5 ~AaY 0~
~uoc~s~ul tissue cul~u~ h~lv~ b~ O.ovul~p6d
whlch r~ault ~n long~ t~l~ ce~l lln~ d~ri~ from
~plAn~ omo o~ t~e a~ lnl~ ar~ hflbitu~
o no~ r~ plarlt hor~non~ ~g ~s~pplQm~n~ ~o
~h~ nu~ri~ u~.tu~- mcd~ ox lonq-t~ n~en~nG~.
Th~ ~.ll lin~ s~Abli~h-d ~ w~ o~ ~h~ D~¢tho~ th~
~r~s~nt lnver tion aro ~p~bl ~ o~ ~ro~lu~ taxol, ~n~
t~xol-r~l~t4t~ aola~oundc. ~h~uq~ ~ell lln~ 02~ rapi41y
~proauo~blR, cOr~lnuou~ly~ All~bl~ ~bura~ ~or
productlon o~ taxol ans!l t~Axol-r~lat~d ~C~mpoun~
~roduct~n ~n~y ~e ~cal11d tO aoyD~ltaroiA~ loy U~ 0
th8 CQll l~n-~- ln l~io2~ tor~. ~n ~ddltlon to ~a~nln~
:
-- 20691~2
5 ~
~ om~?o~ on~ ntly ~r~ ly @~ d ~r~m
Y~ew ca~po~l~lon~ ~xhl~i~ln~ t~ llk~
vl~y, I~-Y~ b~ d ~ro~ ~xtr~ o~ th~ ~a
c~ n~o, or~lnç7 n~w h~ ono ~!or C~ 0~ p3t~ C
t ~v~ n~.
cy~otoxlc dlk~n~ vl~h A~onot~t~
~nt~ln~o~la~ti~ aatlvity ag~ln~ ol~ tu~ d
1aU~C~D~C c~ll llnQs, 4nd ac'ctvlty a~ a ~h~moth~r~la
ag~ ln ollnlc~l t~ial~. Taxol i~ ~ only ~l~me ~rodu¢t
10 ~on~ly XnOWI ~co prQ~o~ a~ y o~ ~aro~ubul~
a~ ~o ln~bl~ th4 t~bul ~ n ~ ~ly ~oc~ . Thl~
~ec~anl~m ~u~g~t~ ~h~t ~A~ol iu ~ pro~oty~ n~w
ola~ of oh~m~tha~ap~aela aq~rlt~ e~at ~tt~ek o~n~ ro~th
hle ~a~i lon. ~or ~U~pO~I; o~ ln~n$10n ~ d
h~r~ln, ~t~Xbl~ n~ral~y ~ nt to l~olud~ aompound-
h~h a~- ~tructurally rol~t~ to t~xol aT~ do~lnad
to ln~u~ r~ uro~r~ o~ taxol 1R ~ioah~ aal ~t~w~yo
wh~ah ~r~ G~bl~ o~ ~r~ducln~ ~xol, to lnt~ t~ o~
tho~ yo, ~nd ~o ~ lYn~ d ~ S~y
2~ ~nt~olito~ e~r t~lxol ~a~halo~annlr~ aa~ln). "~xol~
llX~ act~vlty" ~ r~ to ~o~o~t~on~ whl~h ~h~w ~o~tlY~
r~-ult~ in ~ on~ t~l~t ~ dlctlva of ~
~hQr~othsrapy: a ~ otubul~ stabil~lny ~a~y, aan~r
mcd~ y, or 411nlaal ~rl~
By co~lnin~ ~a~o not ~vlcu~ly ~o~ln~ t:u p~o~u~0
~uo ~ulturo~ and ~y u~lng nev ~ n~ cQ~ ltion~
ob~ul lo~ term ~rowth h~ n nchlclv~ld, an~
inc~ aell lin~ ~rodua~d. A IA~thod for ~t~ hlng
a c~lltur~l oS` ~all~ d~rl~d ~x~ ~e q~ml~ ~xu~ h~ n
u~ao~ul in p~o~ualng ~oll lin~a ~ eor~t~ tax~l and
t~xol-r~u~o~ co~ound~ into t~n ~lt~r~ n~v~on~en~
c~ o~ th~ ultur~ hoa aro n~ ~llow-:
1. 8~1ect 6n ~pproprlAt~ t~u~ lourc~r ~n 4~nt
~hlc:h Wlll ~ u~ed to initlat~ ~h~ a~ ur~.
.
3 5~h4 g~n4r~ u~ u~uro~ 1~ pl~th~ g~nu~
T~xu8, Yar~ou~ ~2cl~ ~xolg whlah thXol T~Ay b~ l~ol~t~d
lnalud~ . Ps~ol~ t~ b~¢oAt~ . au~tdat~
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2~6~122
6 ~
~:~n~donsl~ ~, C~pltata, T. m~O~ . D~n~ rm~ nA 5~.
m~d~ cv. Rlak~li. Pr~ r~o~ re~ ~o~ ~xpl~nts u~ed
- 'co p~oduce tPXol-- ~xpI:~o~ln9~ CUltl~ A~O t~ i plAntE~
~r~ whlah t~ unt ~ t~x~l thAt m~ty b~ ~xtr~ot4d
d~r~ctly ila hi~h uhon comp~r~d ~ u~ xtrac:~d 4r~n
oth~ 0Ci6~1 by co~}lpar~ t~ da gro~ oo~pa~g~ble
a~our~ 0~ 6t~rti~ m~r~Ql~ By th~t arlti~r~on, T.
~di~ or~ rtlcul~rly ~ d~ C~. H1Q~ I fflOr~
pr~t~!rr~d ~oura~ n ~. ~ovl~oI ~ta .
~arX ha~ boan th~ u~u~l ~ou~c~ scol, an~ nu~ s
and roots al~ Ar~ o~ ~x~r~6~1ng ~Xo~ w~x,
1~ W~ tn~ t~t ~ p~ u~ 4~urc~ o~ n~
1~ a 6t~, roQr~ p~rtloul~rly a youn~, ~rQ~h~ ~ccul~nt
4t~3. Y~ at~Y ~ tho~ tn~ ~p~r 8~ tho b~nn~ny
n~ a new q~owth se~on ~o~ ths pl~n~ tur~ ~t~
~ctlon~ o~ ~xu~ m~dl~ cv. Hiok4ii ur~ p~rticul~rl y
~r~rr~d b~&a~a ~t~ r)P ~ d~a pl~nt~ con~sln
h~hor lev~l- o~ ~oxal ~han rax~o ~7r~oll~ ~nd ot~r
p~ o. Explarlt~ D~ ~out ô.~-0.5 hUR ~n dla~tor
~o ar~ it~bl~ .
a. C~lLtur~ t~ nt i~ cAll~lo-~ndual~ du~ to
~or~ calll~6.
Ono o~ th~ con~n~t~on~ o~ ~o~r~ d lng to
~UoOI~Sl~U,!l ~AXW ouït~ Y lnolu~ U~8b 0~ Sl ~sallul~-
2~ induain5~ dlu~ in wnl~h to ~r~w ~ho ~lant.
~ o Ior~ Qn s~l~rlt, ~ ti~u~ ob~a~n~d ~ro3n th~ 3plant
prca~ss~ y sur~c~ ~t~ 2al:i~n nnd ~atl~no~ und~
~sap'ci~ c~ndlt10n3. ~eotloning 1~ aaoo~pll~h~d by au~n~
a s~ lnto plea~ of o.~proxl~at~ly 8-d ~ ln lsng~h, ~r~m
im~aturo 6t~ th~ ~rR elpproxi~ately 3-5 ~m in diA~Dt~r,
and trans~err1ns~ the ~ctl~n~ to ~ u~ oult~
~ns~lly, ~t~ di~h~-, w~n c~llu~-~nduo~n~ ~flUt~lant
~Q~liQ. 'rhl4 1~ dlU~ ~hloh ~tl es~le o~ lrtlt~s~-~n~ AnCI
E~romotinU the ~o~mation o~ a~ gro~th Pr~a~ ~ ox3~l~n~.
3~ ~ p~e~erre~d me~ ~bor51'~ B5 ~s~ tGnn~or~ ot ~l.,
1~68) ~u~ onteo~ wlth pl~nt ~o~n~ uch Al~ ~,4^D ~n~
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L~ YL). I ~y~ , J _ 1 J_ I IJ U U IJ~J ~ 0_ U~ / ~ J
~ L I ~ L ~ U r ~ V L C I O~ I. A rl V l ~ C r~ r ~l A n ~ y ~1 4 1 .S J ~
2V69122
klnetin ~lth~r ~lo~ r ln ~ ina~ion. Ils~lum o~
Mur~hlg~ ~nd SXoog (~ura~hi~, T. ~nd F. 8X~o~, 19~2~ la
~1JO ~ bl~ r t;~ pU~p~ hn~l ~Ay ~ u~ menl:odl.
n~ a~Qnt~ ~uch a~ G~l~it~ ~gcott Il~b~r~ ri~
WArW1Ck, R.I.~ 0~ r ~Y~ ~lx~d ~rlth th~ r,ut~l~r,t m~
to for~ a ~olid ~upport ~n ~h~ aulturo V08l6~ or
malnt~n~n~ ~n~ ~rowth o~ ~hs 4x~1~n~.
3 . ~ubcul~r1 hq ~ol~tod ~e~or~ llu~ a~
lnt~Al~ whi~h ~romOt~ ~ub~lon~ g~o~h.
Callu~ ~or~tl~n io d~3~0~1r~d wh~ obulilr
~ggr2g~t~ of undl~Qr~n~lated ~ re~ rvAd, Tni~
g~norally o~Cl1~8 on go~ o~ th~ oxpl~nts within ~ou~ ~-4
w~ok~. ~uboulturl~g ini~l~lly 1~ ~r~ fl at ~ w~
lntQrvala, but msy bQ ~ rod a~ a fu~tlon ~ cult~r4
l 5 gr~wth .
sl~cting a ~ubQlon~ to ~V~ ll lln~.
!
A~ lh;uort~ t Ptq~ ln ~r~einq o{~ll 11n~ r~
c:2lyabla of ~rod~inç~ ~axol 1~ a~ o~ln~ ~h~ ~llua ~plo
ts ~olcot ~o~ subc~lturin~ to ~vslop ~ olon* o~ th~
ao callu~ Physical ~lection ~ltaria hav~ n d,~v~lop~d
as ~n n~peat ot th~ pro~nt lnv~ntlon. C~llu~ aue wlth
red ~ Drin~ snoul~ not ~a oRleot~d ~aau~a th4 r~d c~lor
ln~i~Atea tnQ pr~tnao o~ p~noll~ ~xuAat~o whiu~ inhlbi~
a~llu~ ~rowth. ~11U# ~oction~ ~xbibi~in~ ~ y~llow calor
2s ~ r~era~ly ~lact~ ~or ~uboultur~n~ ure~
~rowing vell ~n th~ d~rk w~ll b3 gr~n. Fri~ ty 119
~notn~ al~tlon ~rle.rlor. ~ le'Larm~n~d by
o~sr~ng ~ cru~bly, ~r~glle~ t~ turs2 o~ the c~.llue.
Ano~er ~Actor ~1ch indic~ts~ o~m~lea 1:~A1; ~ llX~ly to
r~ t ln ~uc~G~ul c~3ll lln~ r ~U:IepO4~ o
praa~nt lnv~ntlon l~ r~lst~ ~h rat~ a~ the uallu~
aell~. Ini~ially, al~ c~llu~ lo~ q~w~ t~king
ab~ut 2~4 mc~nth~ to ~oubl0~ ~r~ ~r~Able ~9~u~1~n4~ ti~o~ ~r~
.~ f r~ abaut S-16 do.ys . 8ubsu~urin~ wa~ p~ri~ mD~
;
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v ~ r ,-l ~ v L L l ~ L~~ Lr~ r~nr~A- ~ ~ 1 4 ~ I S U ~
2~S9122
ln~:~rval~ out a-6 or~ s ~ ~lthou~h ~ y
b~ l~h~rter o~ lonç~r d~n~lln5J Dn ~ch~ ~alth o~ th~
CAllU~ lnCI~ orlt~d by ~lor, ~d by th~s ~12d~ of th
oall~a ~t~ growth,
s ~y ~rLa~ ~ub~ul~cu~lng n~ c~llu~ ollos~llng
the~o crlt~rl~, r~ln~ ly hom~n~ou~ n~ w1~h
rA~rd to th~a~ crits~ ,, d~v~ chough ~ tlm~
bRtor~on ~n ~x~l~,nt bolng ~ d ln cul~ur~ ~nd th:3
appo.a~anca o~ h~lonQ~ varl~ ome o~ th~ lnt~rval~ ~r~
O m~s~ d i~l yQ~
. ~ulturin~ ~ne c~ll lina on a ~ ld ~ rt ln
~a~nten~na~ ~edi~D~
~ so ~tl~r ~np~r~ant fAc:tora P~ ~ucc~ xu~s
cultur~ ~re a ~ol~d ~uppor~ ~n th~ ~or~ oP a ~3Qbr~n~
lB r~t, and ~naintonanc~ ~dla ~or l~n~ m aulture ~rawth~
2h~ opti~ ul~u~a clondl~lDn~ ~or t~ aot~ b~lon~
Ar~ than determln~d by a ~ultiY4s:1ats ~n~ly~1~ o~ ~otors
Whlch n~ likDly to or~wt cultllrn c~ro~thl ~r~ nao a~
~b~eno~ 11gb~, t~r~ur~, p~ d~ up~ t~
2 0 ~ ncludln~ plant hor~on~ nns~ ~gllro . f3~a~ h~ n
us~l by thoc~ o~ ~k~ll ln th~ ~rt a,e ~ gdr ln nutr1~n~
dlum for ~ AB CUltUr~l~. Unexp~ a~ly, ~ruato~3 An~
gluao~8 ~re u3~d ~or th~ pr~nt lnYontit~n t~ ~nhanco
. oultur~ ~obrth, W1th or wi~ho~ U~ro~a. ~1~3o
~ur~ri~lngly, high~r conc~ntr~tion~ t~An pr~viou~ly
roco~ndQd ~r auaro~ ~f u~d a~ a ~nql~ ~u~ Y~re
~ound ~o ~nhan6e cultu~c~ h conc~ntrst1ona o~ ~ther
gl~co~s or ~uc'co~s ~ d ~ln~ o gavo oxo~ nt
ro~ul~o. Nona o~ th~o U~Ar pro~a~lB h~d b~n x~or~o~
ror !r~xt~
ORtl~u~ cUl~urs ~int~n~n~ or3 lnalud~sd uP~ o~
~rn~ rt to s~Upport ~ ae11~ ~ Thl~ ctruc~
11itate~d ~ub~ultur1n~, ~oll~otion o~ r~aat~, ~nA
~or~atlon o~ C~ll ou~pon~1o~. A~ t~ Ult~
growt21 1B ~C~ ev~d, the C~11 llnelg are ]C~ in ~aln~n~m
~di~m, Par ~c~me o~ tho C~ll lln~ bl~ t~on oe~:ur~,
~ Y L.~ ~ U -~ I . I ' J -- ~ V V I ~ I I J I * ~ 0 4 ~ J I ~ t ( J
2~69~22
o g
~hat 1~, th~ oult~ r~ msin'c~1ne~ wltho~ hq ne~d ~or
h~ nal 4uppl~n~6,
ln addi~:lon to ~llu~ c~ll lln~, c~ u~n~i~n
O~l'cure3 ~r~ ln~ond ~rom we~ ttlbli,~hlad Dt~le c~llu~
cul llr~a ~ d~orib~d ~bc~v~ br tr~ rring ;hn ~noculu~
of C~110 ~2-3 grAm~) o~ ~rl~blH o~llu~ alu~ into ~ d
~dlu~ cont~in~d ln ~ ~ey~r ~ X~ n~r~lly 250 ~1
~1R~ ao~ta~nln~ 140 T~1 o~ e~1~) . F~k~ ar~ pla~d
on ~ ro~y shaker ~cnown to tho~e ~ ~X~ll in the ~rt an~
opo~ d ~ out 125 rp~.
An impo2~tant u~ o~ thb ~w ~ell l~n~ to prodw@
tnxol ~nd t~xol-r~la~ed compound~. C~l~u~ ~nd ~u~p~ion
b~ n~ Of ~axuB d~ lop~ ~y t~e m~ 3 o~ th~
~rR~nt iJ~Y~ntion pro~u~o t~xol, ~s~s~rl~r~ ~nd
int~nnadl~te~ o~ a bl~h~mla~ thway rs~ in~ ~n tax~l
pr~dUCt~on, ~nc1udlnsJ O~!;phalon)~nino and ~acca'cln~ ~n~
a~clva d~riv~ r~3 oi~ ~xol. J~o~h l~lotic ancl y~blot~c~
ltors, proour~or ffi~ nd oth~r ~l~c~thtior~
~ochn~q~ r-a us~ o anh~naa c~c~o3ldary ~otsbollt
- 2~ ~rodu~t~n. ~?~x~l, and taxol-r~latQd aompaund~, ar~
oxtra~tad ~ro~n ~llu~ u~n~i~n ~u~ r~ ana oul~
mediu~, An ~ot~V~ o~n~o~t1on ia on~ ~t ~8 c~abla
o~ lnq ~O~ Y~ U~ ln a ~l~r~tubul~ ~tabill~in~
a~ay, a ~Pncer ~od~l a~y, a cll~o~ x ~ny
~5 com~n~tlon o~ t2~ t~st~ TheK~ aoll lin~ ~r- ~
continuous ~o~rc~ b~au~ thoy ~a~ :be ~ntnined
lnds~n~t~ly, ~3 hav~ noW ~Qn m~intaln~d for S~ y~ar~,
und~r ~pti~ ~on~ltlon~ and rl~t~in ~ lr ab~ y ~o
produ~ t~xo~, ~ot~ro ~r~ n~ p~a~o~s~ o~
~h~y ~row well in l~rg~ wal~ b~or~tor~ th~ a~
oan ~ ~ryopr430rv~ ol prC~duo~on l.bill~g
r~tain~ t~ crlfopr~r~ation or ~to~ 10-15'~
e~ ln~ ro ~ult~bl~ oUXGO~ to ~roW ln
biortl~oto~D to p~oduco la~o quan~lti~o ol~ ~hXOl ~ ~nd
3~ t~xol-x~la~s:d oc~m~ound~. ~o~ h~ ~po~tl~n~ ~r~
rOund ln th~ aultu~ m an~ y b~ obt~n~a by
~olloatln~`th~ ~dlum. Ut~ in~ dl~r~nt ~io~aa~:
~lgna, th~ ~4~0n~ry zn~tclboli~ n~ ln ~lutlan 2~ny
.
~ .
.
~ YLl I ~Y~ U -1 1 . ~ . J _ I ~J_ ' I L~ JV-~ 'J()._ 'J.~
J ~ IL I ~ .lrlll ~ L~ n~ Ln ~ n- ~14~4~
2~63~ ~2
-- 10 --
b~ ~R~ly ~mo~d iro~ th~ Isolu~103~ tlll~lng ~2xc;,h~n~
r~cl~ whi~h QYt~l~Ct only the ~ eoondAry
olit~ ~rcm th~ ~Dlution ~nd no~ tho ~d.la
8 r~ ?b~ c~u~ b~:~ . Ot~r
~o~po~tl~ne~ ~r~ produo~d ~r~d r~tained lntr~c~llulnrly ~nd
mu~t b~ axtr~otod from tt~
~hQ ~owth o~ T~xu~-d~ c~ in~ ln blor~otor~
on~blR~ tho production o~ ~uch l~r~ ~Rnti~ n ar~
~r~n~y ~as~bla. ~hll~ 2;roC~ nt)t only ~ o aE a
0 ret~w~bl~ ra~U~ce o~ chsurlo~h~ra~¢ut~ g~nt~ bue ~ o
saY~A ~be larg~ n~er oP ~X~ 'rhat ~r~ ntly bolng
n~v30ta~ to le~olat~ th~ ~o~a~o~an~.
~ha ~n~t~od~ an~ co~po~ n~ o~ thi- px~nt inventlon
~acill~ht~ new ~ro~n~ng ts~t~ to ~l~t~mln~ whl~h 13~p61C3.~D
Rnd ~rlst~ of ~9XU~ ~ra am~ l4 ~o lc~ng-term, ~tablQ
~ult~lr~ nd whlch Raur~:g~ ~r~ lik~ly to y~ ~ld ~hxtsl an~
- ~xol-r~l~t~d c~m~und~l. No~ onl~ ~ra r~ ly
ra~rO~u~ , oont~nuc~u#ly Q~ , hec3~lblo llnd
r~n~w~ble ~ourc~ o~ th~ OO~O~ h0 ~rov~d, but ~yu
o to ~ac~ thd UnXn~ r~tlon~ in taxol~r~la~o~
compoundsl, ~omo v~iatio~ not obtA~n~bl~ rrDm th~ urQ.l
~ourt~, 9~0 al~o provla~ o tr~ tlt~ Dy ~ ~v~n
~ore pot~nt ch~motha~ pout~a P~ge~llt3 th~ln I~Bve b~4n
l~orted.
Pra~louely u~nown a~pon~nt~ o ~ au~tus~
coll extra~t~ w~th taxol-llk~ ~ot~ity h~ve b~on ~lUtQd
a~ lcz by HPLC ~h~ gh pro~ uid chro~toqra~hs~
.~ The~o p~aX~ co~t~n rA~t~ dhi~h ar~ poslt~ ln
mlcrotubuls~ 2ins~ y~ ~oa~ct~on
~0 o~ o~ltlon~ wlt~ t~xol~ ctlvity ro~ ~ulturot~,
c:ompoa~t~On~ tha~ m~y not b~ lpr~n~ ln n~t~ ourc~,
#Ixpan~s~ th~ horl zonr~ ~or d.e~relo~mont of otlomoth~ra~
~q~nt~. Al~a, b~u~- call lln~ ~lY~er in their }~C!
~ ~ ~Y~ r~l~t~ to tho~ co~7o~ltlona, ~sll c~lltur~
o~r~ to ~A A u~e~ul ~oyr~ o~ n~w ~r~msnt ag~t~,
~no oel~ lino~ di~ar ~n ~ h~unt and natur~ of
taxol ~nd ~axol~ tod Compour~d~ oy o~,ue~
oul~ure undqr a d~inoi ~t oP 0nvlronm~en'c~1 aond~lon~.
., , .;-:
. : ~. . ,.,: , :., ..................... :
.
2~9122
~ 11 --
Ir~hla V lllu~tr~ d~i f f~r~c~ n~ ~o~ o~ tb4 ooll
l~hg~ witn r~g~rd éo tl~ r~n~ ~f ~r~nt OI ~xol
~ro~u~. C4m~p~nDr~t~ ot ~ ars t~x~ ika ~i~ thc~y ar~
e~bl~ OL` ~ n~ cr~tubul~3 ~nd/or o~ giv~
po~ lve ~ull:~ ln o~ncer ~o~ y~tom~, ~or sax~mpl~,
oxlQ~ty OI ~ toat ~g~3nt: ~o
~ ncther a~t o~ th~ ~r~nt ln-J~n~lon 1~ to ~odua~
nQw T~xU~ cell line~i ~y lndu~lng ~iu~ on~ ta~ion~ o~
iQl~ct~d c011 llns~ ~o lnd.uc~d by irr~ lon ~rQAtm~nt
and ~hcmical mutAg~ins~ ~rh~ ~llu~ an~ oiub~lone ~loation
crltori~ and mQ'chodEi ~o inClu~:~ eRllu~ ~nd ~ t~n cQ
lln~ a~i d~scribQ~l herRln, ~Lr~: UFil!~ to ~o~u~ o~ll lin~
l~rom the ~nu~e~ ~bU~C~. Ul~x~vlost li~ht, ln
par~ioul~r ~-A~ ~n~ e~l ~uta~a~ u~h Q~ ~hy~
lS ma~h~n~ ~ul~on~, ~re ~llt~b~ ~u~qQn~.
Anoth~ a~ t o~ p~ant inv~nt~on 1~ ~o
r~ n~ate T~ plant~ ~rom ~n ~ rv ~ultur~s. F~r ths
rc~n~r~tion o~ t~xol-~o~ ing plantl~, o~ a~llt~r~d
~n a higll a~l~lnsoytokinin xati~ undsr a 16 h~ur r~gl~ was
ln~u~d ~o ur~or~o o~g~n~n~ y ~o~ially d~r~a~lng
eh~ o,uxln: ~ytolc~nin ra~lo ~to ~P ~a-~ sop~nt~nyl~nln~
2 - 1~ mg~l in com~n~t~on wi~ ~A (ln~ol~-3-bu~rlo
~cld) 0.1 ~ 2 . 0 ~ o~ ~X~pl~ . 5hoo~ odu~od ~y
tbe~ llus wbr~ th~n ex~ d 4~t~ a~11y ~pproxim~ly
at ~ho ~t~a ~ 1 om ~hoot~) ~n~ pl~a~d on ~org~ B~5
ba~Al medium ~uppl~ tnd ~ 20 qlfl suara~ to lnduc~
r~o~ ~orlution. W~ll root~d ~noQt~a Ar-3 ~h~n tran~rr~d
on~o ~Qrll~ tro-mix 200 ~o~lng ~odlu~, ~a~ t~d An~
tr~n~ rr~d t~ th~ qrQ~nho~ to ~v~lc>p lnto ~ a.
~2uenar~ r. of plAn~o tro~ ~u~tu~ wlth ino~ae~d
produo~ion o~ tAxol pro~ lan~ souroe~ th~t ~-r~
~n~ o~ p~od~ qre~ r ~4~n~ tl-xol And t~xol-
r~ o~poun~ th~n natural plu~ An pro~u~.
gC~'iVl~lD of ~h~ inv~ntl~n ~nalud~ ~rowing O~lU~:
3~ ~nd ~u~p~tn~lon ~ltur~ to ~roduce ~t~blo ~
lin~ ich ~ro aa~ oS proau~ng ~axol ~n~ ~'ch~ ~xol-
relatod ~om~oun~ olo-~d h~in. ~ aell lln~6 ~r~
u~aiul a~ mater3Al ~or ~o p~oductl~n o~ taxol And t~xo~-
: ~ ;
J _ I ~ . J ~J ~ J - I I t l_~
m ~ L ~L ~ rlr, I VL~I~Lt~nul~cn ~,nw~n-~ ~14~41~
2~69122
r~la~od co~pound~ in ~ r~o~r~, r~ ~x~dplæ, l~ orat~y
ec~l~ bior~ct~r~. ~orn~h~ro~ CR10 ~or~aaot~r~ aro ~l~o
aon~w~pl~t~d ~n OY~br ~ rcldu~s4 com31l~rc~a~ n~l~io~ o~
t~xol, aotlv~ der~vn~ o~ ~axol, c~h~ nnln~,
S ~Aoo~ ln, ~nd okh~r t~:~ol ~ r~ r~ ~nd d~rl~rativ~,
guantiti~e ~hat are re~uiræ~ ~or ~n~;30r ~e~a~ah an~
tb~rapy. ~w co~o~ltions w~th t~xol llk~ ~ativi'cy m~y
b~ ob~ n~d ~ro~ o~ tr~ct~ or ~l~uo oultu~ .
~ultur~s Are u~e~l to ~cr~n ~clXU4 ~ Oi6~5;1 Qnd ~ell lln~s
1~ ~o~ ~ro~luc~lon o~ taxol. N~w t~xol-llk~ c~ ound~ ~ay b4
roun~ ln ~ultur~-.
D~Oll~
t,ho ~ro~ gr~du~l tr~n~sr o~ In Ylt~o
~en~rat~ ~lant~ ~ thA ot~ lo ~nvlron~nt ln s~rdDr to
lS ~ d ~udden d~sloc~lon du~ ~o ox~s~v~2 wat4r 1~
~Q~S ap~ara~wY iEo~ gr~wln5~ ne6 ~n ~ lar~e
~cal~
A.~rll~t: Ronte~ ~cty~ irlit blo~otor~
~raun Bl ch (~ 'ca~ ~ir 11~ blor~aator~
~Q LH ~ ntation ~00 ~erl~)
~tlrr~: N~S~ Bxun~wlok (C~llla~n & B~o~lo~
F~ nt~t ~ on
~orf`u~lon: N~w B~un~wlck
~embrAnG-~low~ nut~nt ~oduD~ ~low~ ov~r a ~mbrAt~
. 25 Wn~h ~upp~rt~ t~e c611~, ~h~ u~ 1~ r~r~ulat~d.
OthQ~ no~ l de~l~n~s ~r~ ~u1~4bl~
~aJ,.lla~ an ag~req~t~ o~ d~i~r~ntiat~d cell~.
n ~t~o or lr~ v~,vo ~yfa~o~ wh4~rolr~
a r~ult 1~ produa~d ~y tout ~n~ ld r~ult
dat~r3~1n~ 'co be pr~dlctlv~ o~ ~llnlo~ ec~o ~ the
' Ag~n~.
aOmPO~ttiOn ~r livln~ asll~ ~nalnta~r~eCI Yn
~1 ~o .
~L~: g~oup of 4~ ln YltX`o ~or whiah Jro~
charact~gtice ~Yo ~cor~ ~t~blo un~r ~ ~efineA ~o~ o~
env~onmant~l condl~i~n~, and ~hl~ o di~r~rontl~t~a ~o~
o~r ~ n~ h~ rlo~o~,
- , ; . ~.
.... . . .
- 13 .v 2 ~ ~91 22
~o~1~vlon o~ livlnq ~Q~l~ thRt nræ not
~tt~d tc ~ ~ol~d ~uh~tr~t~ but r~th0r sr~ ~u~p~ ln
a llgul~.
ClU~ S qlo~ul~r or no~ul~r 4~r~t~ o~ a~llN
ob~ ra~ n th~ s~urfaco o~ ~ a~
~n~ d~um ln whl~h oultur~d oell~
~qr~t~ into t~ m~lurd ~JArlou3 m~t~ollt~ an~ other
aompon~n~ c~ ro~lrth and, dlvl~lbn.
h~r~n ~o ~ ch~ r~to ~t ~ h
o cell~ ln cultvura doubl4 by ml~o~ doubllng rat~.
111 ~ t~chnigu~ appll~Dd to ~ o~ll
cul~u~ wt~ch ~nar~o th~ y~ld o~ ~xol Ox ~xol-r~ te~
co~poun~o th~ ~r~ produ~d by th3 cultur~ ~h~ h~
o~ t}~ iria ~ at m4y bo ~mnunolo~ 1, t2~t 1~, th~
lS coll~ m~y ~ount ~ dQ~n~ aga~n~'c forelqn ~ant~- by
produclng taxol .
BlotlQ: ~x~mpl~: lnalud~ aut~cl~ d gunu~l o~ll w~ll
~rllgmen~a ~nd s~or~.
c~tl~ t ~x~plo~ ls~e: lud~ ~alt~ orh h~
m~t~ ,vanadat~ d ~nd m~rcury.
F~J~ty: ~ra~ ru~bly t~ a oonsi~t~noy,
}~b~u~ a~ c~ n vlt~ wnlel~ e.r~ m~lnt~ ~n~à
~y ~u~ul~r~ ~nd do n~t r~ n~trlont modlum
s~ppl em~n~ed wlth honnon~ for ~int~nan~; th~ ltur~
~rQ usually char~ct~le~d by rolatiYoly rapld ~rowtl~
rat~-
~s condltlon wher~lr a ~ n~ np~bl~ o~
balng ~ intaln~ by ~r~al ~u~:ul~r~ v~thout th~ u~4 of
honllcna~ upplem~nt~ to th~ nutri~nt In~dlu~ in o~hl~h
tt~ cell~ aro ~in~ln~d.
~: llt~r~lly l)ln S111~8E-~7, an ~ la.l on-~lr~n~hDnt
wh~ah ~l~ia~ th~ ln ~vo aonq~tlon~ tanarlc~ ~nCI~
~nnrally, ~rowth and r~o~ual:lon o~ ~lolo~ie~l
Illl~tC~ B .
3~ a nutri~nt ~om~o~ibn ~or tn~ ~rowth o~ l~vin~
~nat~ u~h aa cell~, til~U~ fflbry4~ d th~
R~ lon t~ t~mo ~t wh~ch. ~ giY~n ~onlpounfl will elu~
~ B cr~romatogr~p~ lu~n ooun~ln~ fro~a th~ s ~t
~ YIJ~ I t I . I . .~ J ' U~' ' ~IU'I ~,J~ 1 't ' '' I '_' t-J
_14 20~9122
.rhioh ~ ~mpl~ w~ in~r~uoa~l o~o tho aolu~n.
_: ~ m~buli~ thht L~ no~ p~
~r~ry ~a~c~b~ n~twork o~ th~ c~ cond~y
~aht~oll'e~ ~r~ u~ua~ rQ~nt u~ly in c~ t
~p~al~e ~n ~om~ w~y (~.~. d~n~
t any ~t~ln~ th~t c~ul~ b~ u~ to
~ulture ~ell~ on a ro~ry ~AX~ O.g. ~rl~ 0y~r r1a~
~r5[lDs~: ~'orcln~ ~bubblin~) alr throuyh ~ gu1d m~dlu~,
:~: cor~dll:lon o~' c6~11 aul~ S~owth
wher~ln dou~l~ng ~m~ n~ ~h~ ~ul~l~r~ ~V~ r~aahad an
Q~aU~ librium und~ a de~ln~ t o~ cull:ure ~on~tlon~ .
5u~ cultura ~r1~ ro~ lo o~ c~ r~
a par~nt oultur~.
~ n a~ll aul~ur~ c~o~ltlon ~rlY~d ~rom a
t 5 ~ampl~ ~2' cultur~d cell~ 1 a~m~ b~lng tr~ rr~d
eo a dlfr~rent tl~u~ cultur~ v~n~l ~han thl~ aont~lnin~
~b-. aulture o~ o~llA ~rom ~hloh th~ o~ lnea.
Tl~uo c~l~r~: compn~lon o~ l~vln~ u~ ~lnt~ln~d
~ Y~er~, th~ t~ o~ll cUltu~ nd ~s~u~ cultur~ ar~
o~tan u~ad ~n~*r~h~ngo~bl5~ y ulll ~ hor~ln ~o
r~er t~ ~ny a~o~ition wh~r~ ~ pl~r~llty o~ r~
~ln~ain~l In vltro.
~R~F n~A~
'~ oth~ o~i~ct~ Imd ~dv~n~ n o~ tha ~nv~ntlon wll~
~5 b~ne ~ r~nt upon raa~n~ th~ ~ollowln~ dot~tl~d
d~crl~tlon ~nd u~on re~r~n~ to ~h~ ~a~ ln wh~t:h:
FIG. 1. 8t~m ~ an~ ~hc~ln~ callu~ ~orm~lt~c~n A~t~r 3
w61ok~ in cu1turs~.
FIC. a. Coll ~u~*~ on~ rat1ng pr6l~r~ yellow
3~ c~ ko t2~ le~t, ~n~ r~d c~ whioh ar3 to b~ ~voided,
to th~ rigl~.
F~G. 3. ~ad br~wn o~llu~ ~re ~ ~ at th~ top, pr~ d
y~llow cal1u- at thr, bc~ttor~.
:
. . . .
,~
V U 7 ._ V_. .J _ ~
L ~ - J ~ J ~ r ~ Lhnu~ ,n~An~ ~ l D,Y~4 1~
~069122
-- 15 ~
FI~. 4, A ~ul~ure o~ oallu~ lin~ C~1 b~ brlln~ r~rt~;
two raPte ~r~ ~hown, ons ~ 4 w~ek growth ~orl~
t) an~ th~ oth~r ~t~ ~ 7 w~ rowt2~ ~srlod fri~ht),
E~ah or ~h~ rn~ W~ lnl~lallY ~ rith 3 ~ 0
oallu~.
PIG. 5. ~n vltro ~lcrotubule ~nbly and ~t~hillzin~
~atl~lty ~!leDIonatrntDd ~y t~x~ ol~od ~rom ooll li-~o
CR~ ur~d by dar)t-~lel~ mloro~eopy (to~ ~ot
T~Q cont~ol t~ot~om ~ ~tur~ y~ ln
~Danner wit~out ~he ad~ltion o~ taxol,
`:
FI~. 6 . ~E>I,C ~ro~ f coll ~xtr~ how~ng sc~l~
~ak~ at 18 ~nd a5 ~lnu~ 81~ltlon. 1~ pro~ile to th~
le~t l~how~ au~hon~lc taxol, ~2~ mann~n~ nd ~Aa~at~n,
~h~ pro~le to thQ 5~1ght ~l~o lncl~ r~ultli o~ ~n
~xtrElct o~ &~11 lin~ c~-~. PG~X~ 4 and 5, at 1~ ~nd a~
minut~ ~lu~:lon, ~rer~ po~lt~v~ ~e ~i~rotubul~-
~t~billslnq ~q~y (~o ~l~t~rl~ nd r~
~2TA~D D~CR~ 0~1
Ualng thc ~n~th~ o~ t~e p~ nt ~n~r~ntlon, succc~
~û in pro~uolnq st~5bl~, long-t~ n~l8 ha~ bo~n
aahl~v~. The ~ollowlny ~c~inati~n~ o~ st~p~ ~re n,~
~o~ ~reA~lon o~ xU~ C;Gll lin~s.
Tll~ cal~ Rnd ~oll 6u~lonq o~ th4 pr~nt
~ nvont I on arQ indua~d ~ro~ th~ a ~p~cle3 lnolu~inq
2~ T. br6v~011~, T. b~cc~t~ pAnd~n~ p~d~t~
~dl~ cY. D~n~i~P~i0, ~nd ~ dl~ ov. I~io~
Young ~t~ xplant~ ~r~ pr~ red a~ a tlu~u~ ~urc~
to lnduc~ callu3. Youn~ ~t~ ro thc~ ~h~t ~pp~Ar
n~v qro1lrthe on a ~ pl~t th~ ~t~ gen~rGlly ~r~
a~out 0.~-o.~ d~a~ r. Youn~ Or ~auNJ~ 4lfl
av. Illok~ o p~e~errc~ b~u~4 ~cn~ ,t~ ound
to p~cdua~ hlgher lc~vol~- o~ tax~ n ~th~r ~axu3 o~ole~
lnoludlng th~a ~ts~ oP ~ r~vi~ol~ Al~hough ot~er
plant p~rto ha~ h~ l$ty ~o produc~ callu~ youn~
~5 13tQ1tl~ ro~ id~d t~l¢ b~t ~ ltil on A ~or~ nt ~ ia
`:
J ~ 1 1 , .J . ., ~ o ~ o
2~6~122
- 16
~ ung ~o~ ~xpl~nts Pro~ x~oc~ ~ca~ çad
and ~actlor~d und@r ~uo~tlo aondlt~ on~ atlon~ ~r~s
gxo~n on c~lluai-lndu~in,~ dlum. 6ult~hl~ ~dlu~ inc~lu~
O~nb~rg l ~ B~ ~dt UTP ~Gam~r~ ~t ~ 8) ~l~a~ls ~
fo~ti~i~d witl~ ~mbor~ 5 Ylt~mln~ cDR~ln hyd~ly~te
(1.0 ~Jl), And a ~ain~ln~t~on or ~exuc~o~a~, g1U~3B~ and
t~uc~roe~ ~ot~lllng ~s-40 ~ c)r ~ala~ oe~ n~, hlgh
concs~rltratlon~ of gluao~e~ l~rl~ot~ 3t~ or ~uaro~ 40-~0
i~ pr~ rQ~ to th~ ~tarldl~rd 20-30 ~/1 oP ~uoro~ao ir~
nu~r~n~ a~ ur~rl~ingl~, yl~co6a ~nd/or ~ru~q~
~I~!Iy ~pl~!lGU I~UCrO8e. PlAI~t h~on~ r~ o
~uppl~n~ ths m~dlum a4 pl~nt growt~ r~sgulDto~
;; ~allu~ lncluot~on. A-larin~ y bo us~d ln oomblnAl:10r~ h
1~ kin~tln, th~ latter ~ A r~ngR o U.1-1. 0 ~g/1. In
dQtormlning ratios of ho~morles t~ U~, a hlg~ ~uxlz~:
~ytDklnin r~tio ~ r~fe~red l~o~ a~llu~ lndu~tlo~, Ps~r
~xa~31~, 1 mg~l ~, 4-D to o. 2 r~g~l kls~tln~ 3 ~Jl ~P
n~p~t~al~n~ ~catic~ Mc1d (N~) ~o 1 ms klnotln. With
~eg~ l to Auxlns~ ~ t ~ r~n~ .0~ m~ 2,4-D
~t a ran~ o~ o~l-a~ gJl) o~ IBA (ind~lo bu'c7ri~ AalCl~
a'c ~ re~nS~ of l.~-~.Q ~q/l) a3~ ~uit4bl~ an
illu~tratl~r~ bodlmenk, 1~ 2,4-~ an~ 0.~ p~m 3~ ti~
~o ~d~d to th- culture~ ne or ~n co~ination wlth
~5 Gol~ito or qg~r a~ ~elllng agQnta.
~x~lant~ ar~ plao~ ln tl8~u~ ~ul~ura v~e~,
~an~r~lly p~trl dlsh~ o~nt~lnin~ d1u~ lo~ w~h
B~lr~t~, at ~bou~ ~ 2 q~l oono~ntr~tlon. Prlor to k~in~
~l-pen~d ~n~co ~terll~ pe~rl dld~4ll, th~ :nQdiu~ ~:oy l:~o
~t~rlllzQd, ~or ~cam~ uto~l~vQd at ~a~ o~ about lS
~' nut~
Explant~ are ~n~ub~t~d ~ s~ c~o~apl~t~ d~rlc ~t
t~p~rAtur~ o~ 2bout 23-25 ~ hcll p~l o~ ~he~ ~Qa.it~
pr~ ly ~alnt~ln~d ln ~ r~n~o o~ , 40l~
3~ cultura~3 ~ay pra~er to qrow ln tha ll~htt ~t ox~pl~l,
~o~ hle~ rl~l~ra~con,t li~h~ ~or 1~ hours ln ~ a4 hour
ayc~lQ,
Indu~Q~ c~llu~ ~B ~ g~ ell ~ul~ 1~ lnltl~lly VQry
;, . ~ .. .
': ~ ' ~ ', " ' ',: .
~ 17 ~ 2l~ 691 22
BlOW grr~w~n~ ~n~ prodUoBO ~hsholln QXUdltOO ~OhU01nS~ th~
ca11~o to t:urn s!~fl) whl~h ~r~ dLItrln~Q~n~al to th~ growth
0~ th~ c~l~U~, avsn wn~n ~a ~llU~ lo r~ontly
~u~ul~ur~d. A~ d~arlb~d in m~ro d~t~ h~ t~r~al~
3 s.nd ~l~thod6- ~aa~lon h~1n~ ~b~l~n~4 ~y b~ t~d ~
th~ ~uboultur~ o~ ~ndu~ cnllu~ 5 on ~h~ c:al
~ crlt~ lnoludlnsa comp~r~tiv~a ~rowth ra~, s~s6~n~0 o~
r~ ~olQrlng, ~rQ~enc~ o~ yellow col~in~, and Pri~hlli~y
FIG. ~ I'or c~ u~n~on ~olo~ crlt~xl~; and P'IG.
3 f~r c~llu~ ~olor ~rit~r~
8u~aultureç! s~ d ~r t~ t~ di~lo~d h~r~$~l,
~r~ tran~4~r~ to lol~d 8u~0rt~ erzlbly msr~r~m~
r~ts (~G. 4~ (Si~ h~mic~l ~4.) ~n ~edia ~Uc~
ohowrl in ~I!able I~ ~r~n~ r~f~ w~r~ prsf~rr-~d Par tha
1~ aultu~ o~ c~llu~3 b~cr~u~ thi~ syst~ lar to a
~mam~an~-flow`' typ~ ~or~a~ox. ~y u~ o~ A r~t, ~
aultur~ diu~ han~d ~td ~an~ lat~d ~lly, and t~UB
a~n aao~l~ b~ ad4~t~d to l~rg~ ~c~l~ oultu~a, Oth1~r
r~ on~ ~or u~lng~ raftc arD th~t callu~ t~nd to ~row
ao ~tt~r on ~ne~r~ne r~t~ ~han on agur o~ oth~r 0~11ing
ag~nt~, ~n~ al~a lC 1~ r to h~r~t callu~ both ~or
~naly~ an~ ~ubculturln~y purpo~e~.
Taxol, ~tiY~ d~iv-~tlv~ o~ e~xol, c~phal~nnln~,
l~a~tln and oth~r pl:o~ur~ora A~d l~to~dt~ t~xol
23 ~ x~ od u~lng t~~ Arlllllysll~ by ~o~p~rln~ ~e
r~ on tlm~¢ o~ ~uth~ntlc ~nd~xd~ ~n~ plant ~x~ra~t~.
Thls An~ly~ acco~l~ ~h2~1 by ~raat$ng cnllul-,
~r~ nCI ~IPT,C an~ly~ n tl~ ~xtract~, And ooll~ctin~
40m~n~ntl- of mA~or ~ak~ nl~ ~o~pon~n~ of ~ AIU~
ar~ t4~t~d r~r ~otivity ln m~crotubul~ ~t~bll~21ng ~ ye
(~XG. B, ~atori~llo ~nd ~h~dB) ~and in ~n Y~tro u~na~2
~odsl ~y~ter~ d ~ ~o1De~ he~ n . c~nCer t: Q
su~t~ or ~ar~enlr~ lncludl0 Bl~ M~l~no~na; MX-l ~nia~
x~nogra~t; P3R8 l~uk~ml~; K~7 I,12~0 lou1c3m~.
Tslx~1 D¢tiYlty l~ o aon~ n a~11 lln~ ~y
~o~lng ~a'c ~X~:r~c~ hav- tho ~bilaty tD ~t~blll2
miar~tu~ul~ ~tlY~ty in ~h~ ~m~ r a~ t~xol ~P~a. ~.
Anot~r m~ns o~ idon'c~ t1on o~ t~ acc~mpli~ d
~ ~ U '~ ' J ~ ' U ~ ~./ U -t '_/(- _. U . . ~ t . . '! ~ (j
---'' ' -- ' ' '----'`' "`--'`''`' '` ' `' ~ '~L ~ rlll I V~C I ~ ~ vl~L~ ~vr~hn~
~69~22
. by ex~r~c~tln~ O~llutl ~1311 lin~ pur~yln~ thl~ ~xtrrt~t
by HP~ And ~d~nt$t~n~ t~xol b~,~ util~n~ H~ ~nd a-~ high
~olutl~n nu~le~r m~s~n0tie ~ort~noD (~) ~na~y~i~
(P~lZor~ 1., 1992)
~lr~re~t bloreac~or d~lgno ~ av~ to ~axi~lz~
gro~rtb r~t~8, ~alx~l, aC~1Yd~ d~rlvatlva~ of ~a~ol,
~eph~lom~nnine, bacc~tin ~nd o~ pr~cur~or~ or ~f~xol
mad~ by ~ah o~ th~ o~ll lin~ nd o~or~t~d ~nto aolu~os~
(W~tb~r~ ~nd G11~ 48 J P~ t al., lg~7) . Pl~nt
~0 C~ mo~ z~d in Y8~1~US gul~ Ana ~mbran6l r~n~
ha~o ~n U. ~d in bl~r~to~ IBrode~11u~, 19~5~ .
Alg~n~t~, ~gar, aqaro~4, kh~p~ o~rr~ n~n,
~olyacrylA~id~, polyllr~t~Ano And vAriou~ ran~
~on~i~rat~on~ haLv~ ~B~n us~d to ~antra~ ~lla t~rad~u~,
1~ lg~a ~ ~m~d6rod and sk~k~r~ l9gO) ~ By o~in~iglng
Qn~lr~ nt~l b~d1~ on~ ~unh a~ v~
co~po~ on~/dln~ a~ ~xyg~n, tom~r~tur~, pl~nt
2~ox~onoa, ~H of th~ ~r~wth ~dit~, n~trl~nt~, vl~amin~ an~
r~moval o~ t~x~l fro~ ~h~ dlia, d~ir~d com~ur
O oxtr~:tion ~ilay be n~xlmi2~d .
~hn 1~VQ1~ 0~ t~xol ~ound ln tn~ C~Q11 lin~ o~ th~
pr~nt inv~ntion ara ~ a~t ~ to 2 ~o~d ht~har thæ.n in
~t~ ollV~5 and b~rk og ~axu~ modl3 ~. ~lck~ Ta~ls
I) . ~ha~a l~v~ nay b~ lncr~æed ~th~r~ In ~n~ar~l
~5 s~ varl~ty of tis~u~ ~ulture~ h~ bn~n ~Ihown th~t
c~ltur~o p~nduclng low or ~det~ot~blo lsY~l~ o~ on~
~rodu~t6 Are ~l~vata~ varl-ty b~ ~an~ d~ng
~p~ tl~n o~ culturo ~on~t~onc-, pr~r~or ~a~ln~
of biotlc ~H~ln~ln, lg85t ~F~Icul et al., 199~), an~
~biotlc slioito~ lnclud~nq ~)~e l~o ~ ultr~-v~ olot
lrra~lo.ticn ~X~tu~ch ~n~ tl~n~orf~r, 19~0) ~nd h~
~etal lons (~h~ All ~nd ~hi~ah~ , 19~ l~ nddltlon
or ~r~our~on3 'CO ~1~11 cultu~ OI ~pt~g~um hr~lîor~
h~ beor~ r~3~or~d to ln~A~ pro~uation o~ th~
~n~it~nor co~p~und tri~iollda IMlslawa~ oe ~ 19~5).
Proaursor reedlng~ bio~ici ~n~ tlc 811ai~or~ a~ ~ofin~ld
h~r~ln, Rre sul~b1~ ~or Ul~e with !~tt2n2~ cultl~r~.
The lndu~tion o~ 2lut~tlon~ in oall 1~ by
.
' ' ~ ,` '
- . ~ ~ . ' . ' . .~!
.~.
~ ' '' .
I~ \ U ~ J ~ ) U~ J t '.~0_. IJ._ I ~ . I tO
' n~ ; v ~ r ~ J L ~ L~ '~ UI~ r w~ 14 U S 413 9 ~ 3
~0~912~
_ ~9 _
lrradl~tlon ~nd ch~ hl ~llt~g~n~ ro~ult ~n a~ e~
wh~ v~r~rodU~ e~xol, ~lV~ tv~t~vo~ ~ t~ol,
c~phalom~nnln~ b~ao~tin an~ ot~L~r pr~u~ r~ 0~ tAX~
ln ~d~itior~ cttic~ ~nd ~bl~tlu ~ trra, ~e30di~g Or
S ~ uraorf~, ~nd o'chsr cl ~ citE~tlc~n tochnlq~ ln ah~k~
cultur~ and larg4 ~cale ~lor~Aotoa~ c~upl~d ~ith lar~
wala ~uri~lc:h~lon ~ay $n~r~a~ rodllct yl~ld.
~lant~ ~ay ~ n~ra~ Prom ~ u~ure~ to b~
yet P~noth~ ~oUrG~ o~ 'c4xo~ a~nd, ~or~ in~v~r~lngly,
perh~ o~ ~h~ naw ~und~ id~n~ d ~n ~ltur~ t}~at
have t~xol-like 3~t~vity.
Tne 2~ t.r1n~ ar~ x~tlv~ ~x~mplfa~ o~ ~ p~ent
ln~an~lon.
13 1
p~:avAration ~1~ A ~Lll~i 0~1~ I.in~ m ~xu~
~eW15,t ap~enring, uc~oul~3n~ lcsy) ~tMI~ grow~h Pr~
T~1~8 2~l~u'1a ~v. ~ k~ll w~ harvQ~t~ rQaa loo~t~
~n ~rhe P~nnsylvan~ 6t~to un~r~$ty, ~l~ ity Park
C~mpu3 ln th~ ~r~ng and ~ ly ~ro~ d. Tn~
n~dl~ wcr~ r~move~ 'chQ ~t~ na tho ~tosn~ a-lt ~n~o
3 C5~ ~ctlon~. Th~ youn~ ~uc~ul~nt ~ts~ tl~e~u~ w~o
cte~ b~c~u~ ~t conta~n~ th~ hl~h~t 1~VQ1~ 0~ taxol
ln ~ p~ 8 ~ o~CI. 'rh~ o~lantn w~r4 eur~ao~
~t~rlll2~ fo~ ~0 ~eaon~ nnl an~ ln~edlht~ly
pl~ced ln lot ~ ac~ ~olut~on (~lv) plu~ n ~o for 20
~nuto~, rin~d 3X wlt~ ~taril~ ~At~r and ~otlon~d un~r
n~ptlc ~ndl'clon4. O~ar 0~n~A~rCI ~ur~a~ ~t~rl~ lon
proa~flur~ ~re alllo ~ultnl)l~ aoo ~t~rili~t
~D ~te~ wer~ cut ~nto sl~atlqrl3 undar ~p~l~ conditio~e ~nd
pl~d on GAmborg~ ~5 m~ lu~ an~ ~lnu~ 2,4~, Xinetin
or ~ plu~ Xlnt~eln ln th~ a~ ~rk ~t ~3'a.
Un~iff~r~n~iA~ed o~llu~ e s~ ln~tlally ~lo,r gr~ng,
how~v~dr~ thr~h lnany ~u~culture~ a ~r~a~le y~llowish
3~ a~lor~ ol~ill lln~- d~ n~t~ ~1 with ~t~lbl~ slrow'ch
cha~a~tari~uu w~3 obtaln~ hl4 c~ r~w- on
¢a~bor~'a B5 ~dia (~blo ~:E) ~n tl~o dar~ At a~Dc wlth~ut
v v r ,~l ~ VL C l DL~ c r~ r 7~ J 14 ~ '~4 1 ~ ~ v - ~ 4
~.
2069~ 22
~ 20 --
.:
~h~ ~upply ci~ ~ny ~xo~nou~ l~ox~nonoo .
- q!ha c~ nA Cl ~ int~lin~ ~n ~adlA æu~ nt~d
wlth ~eln h~droly~nt~ oo;~ ~nd ~rUctoD~, wh~
~ound to Dlg~ icant~y ~n~r~ o ~* g~ow~h r~ o~
s c~ol~ lin~ b~L~ IY~ ~ .
~r q~;ll lln~ we~ rl~ r~ h~ c~llu~
aoll llne Gl w~ ~ubclos~ n modl~ g's ~5 ~dl~
~bown in T~ h~ oallu~ o~ll lina os~ theh
t~an~f~rr~d to mentbr~n~ ~a~t~ . 43 cont~intng li~ld
~dl~ ~hown ln T~:Le II at 25'C ln th~ d~rk. ~hi~
l~no W~ ubolon~d ~nd 1l ~11 llne Witll ~t~ble gr~w~
c)~Rr~t~ri~tlca w~ d~ rod (cR13 ~o ~x~1~ 2) .
12X~E.~ ~
9~_
~
pr~ In~r~2n
A ~allu~-d~riv~d o~ll lin~ d~ k~d CR-l, d~ri~d
~om ~ oallu~ .gnate~ ~1/hss ~ho ~ollowlng
~hJIr4~eeria~io~l gro~ ir a~l~Ure~ ,n ~h~ nutxl~n~
ao m~ is ~nl~r~ 5 ~e~dium~ q~o~ in th~ X ~t ~0-
26'C) do~ n~t r~lr~ æ~ nou~ pl~nt ho~non~ a
h~b~t~at~ lin~); RhDw2l ln~rat~ oo~ d t~
lt~ bq~Qlin~ ~y 3ddltion t~ um o~ c~ln hydroly~t~,
glu~e and ~mCto~ to th~ Alu~ e Ta~l~ IIl);
26 ssthi~lt~ ~ d~u~lln~ r~om ~ 1~ d~ysl ~nd ~ w~
~t~ row~h in ~ul~u~ a~ll lln~ wa~ OApi~ a of
- ~ar~t~n~ taxol, ~ctlv~ ~ar~ tiv~ o~ taxol,
~h~lo~arm~n~, bacc~tin ~nd othsr preaur~or~ <~f t~:~cl
into th~a growth m~
~n~ c~ sriYed 0411 11~ dQ~ nAt~d ~Rl p~wdu~od
r~pid ~rowth o~ c~llU~ aft~r ~uboulturlng: ~h~ ~h~
c~llU~ uHe~Qâ on a r~mbrnno ~t ~pro~uc~ 50-70 gr~ o~
ca~ a wl~h~n 7-lo w~oX~ o ~Gulturln~, ~u~ thD
add~tion or ~ro-h melllum. tF~ 4)
3~
" ~_~
: . .
.," : :;
- . : . .. .
. - - , ~ : , .::
..
V~ n~r~ 4 ~ ~r~ l ~ y v l u ~
2~$9122
~.
rwO ~uo~ ion cul~ur~ l~ne~ w~jr~ ~loo ~dtl~d ~y
~:n- m~hs~ oi? ~ ~r~ n~ ln~Qnt~on ~ro~ llu~
llno ~:1. A ~uap~n~lon ~u~ n~ ~3 w~ d~riv~d ~i~h
s~abl~ gr~ ohz~r~c~eri~t~c~ W~n ~oW3~ at as~c ln
d@.rk wltn modifi~ a~mborg~ ~5 ~la ohown in ~ lo XY.
suopen~on oQll l~n~ ~rodu~ in t~ mann~ w~ra
typically ~low ~r~w~nSI, now~v~r, ~ou~h ~u~olor~ln~
lQ c:~ll llne ~3 wa~ a~t~in~d And h~4 ~ 3~od~ra~ ~r~ r~t~.
~hin ~ell l~no ~ odu~o taxol, ~t~ ~r~u~r~ ~nd
~otlv~ derlvat~rQ~ o~ ol 4nd ~ocr~t~ th~ h~la~l~
nto tho sol~ orl and aan ~A~i1Y b~ r~ov~d ~ro~ ~S~
~luti~n U~1 nq ~x~8,n~ r~ln~ .
Anotho~ ~u~p~n~lt)n ~ult~ no d~gn~
W~9 o~taino~ ln a ~i~R W~'y ~ro~ CRl oall~ lin6~ rown
on m~mbrAno ra~t~. A tin~ o~ 3u~n~1On ~ oun~ ~n
th~ nu~rl~nt ~olutlon in which t~ ca~lu~ ~Rl Whf!l gr~w~-g
on mQr~r~n~ r~rt~ . Th~ ~u~ au~.~U~
od~ced u6ing ~tho~ o~ tb~ ~r~ht invant~on wherg~ln
o~lls sl~ughQd o~f a211u~ c~ n~ C~l lh~o ~u~ n
ln th~ nutr~nt ~n~d~, w~r~ qrown ~n 1 ~ slhh~r i~
with a~ltat~on in th~ d~rX ~ ~bout ~4-a~'c ln C~m~or~
~ ~edium ~ 4hown ln ~bl~ ~v. ~h~ ~u~pan~ion oul~ure
wa~; do~ign~t~d SR-l. eu~n~n oultur~ ~R~l w~ ~Ap~bls
~t ~acrRtlng taxDl, ~ctlve d~rlva'ci~ o~ t~xol,
acphalom~nnls~o, b~a~tin ~n~ r proo~roor~ o~ ~Xt~l
lnto ~ ~rowth ~d~.
A ~a~pl~ o~ ~ell~ ~rom call lln~ Gl (EX~L~ hle~
g~ow~ ~n th~ dllrk W/~18 ~ ~rad tc~ ~ llq~t aycle o~ la
h~ur~ ht, 12 h~r~ do.rX, ~t ~'c. Tr~r~H~-~ to tho
llght ln~ tla~ly c~us~d ~ ~lo~ ~o~n in g~owth ~t~
b~own dl~ao~or~tlo", ~o ~v~ld a~ ath ~hl~ cllllu~ lin~
plaa~d on ~ov~r~l hormon~ r~ eont~ln~ng ~,4-D ~nd
; ~ ~
.
,
"
,.",
~ t~yL~ t ~ J ~ I () V ~ ~ IJ~J ~ J.~
2~122
- 2a
klnQt~n. I`hl3sc- aoll l:Ln~ cl~ha~ and ~ ahllu~
c~ 1n~ des1qnat~d L~3 which g~ow~ on Ga~b~rg'a ~ ~edi~
w~ ~el~q~ ITI) ~n ~ ltgl~ 3 r~ul
nou~ plAnt hor~onQs (1 ~m ~,4-D ~nd o.a ~p~ kinu~1n)
in ord~r to ~rosr. ~hl~ ~allu~ lln6~ h~ P~on RcC~ t~d
to ~no~bx~r.o rn~ nd ~u~n~ion oul~ h~v~ b~on
initi~ted. LG3 wa~ ~ubault~a on di~r~nt hor~
~gl~ con~tin~ v~iou~ bln~tlom3 9~'
2~i~0~ntyla~nin~ and in~ ~bu~yric ~ 0.l, ~ .o ~
0 5.0 pp~ h~ ~11 lin~ p~blo o~ ~oer~ting taxol,
V~ ~erlvatlv~3 o5~ taxol, c~l~h~l~mann~ne, ~ c~1n ~nd
oth~r p~c~lr~o~s of t~xol lnto *ho ~ lu~,
~h~ c~llu~ coll lin~ L~3 w~ ~CUl~ d on ~o
dl~ferer~t ho~#Qn~ r~gim~ ytokinl~s~ and ~uxlno lr~ the
~i~ht at ~s ~. ~y u~lng ~h~ thod ~h~t ~o~atlQn w~
irlducsd. rhl~ ~ohn~qu~ w111 b~ lnv~luabla ln
r~Stsneration o~ pl~n~ w~th ~up~lar Itaxol proau~ln~
oapa~ ti~3 ~nd Afl. ~ 1/ oP ~ atin~ taxol l~v~ n
~l~nt~ ~rown und~r A~ptlo cond~tlon~ S~ a
2~ ou~pon~ion cu~eu~e d~3rSv~A ~ro~
~La2~ 5
xol, oo~halo~Q~nnlna ~n~ ~aoc~tlr, ~ra ld~nt1~1~d
~5 in ea~h ~ axw w~dl~ ~v. ~l~k~ t CP.l, ~3,
~Rl, And hG3 u~ln~ HPLC ~rlaly~la ~ t~lri~ d
Mat~oda). 'rhi6 wa~ ac¢o~p~h~d by making~ ~om~arls~ o~
~tent~on ~m~s b~tw~n ~lAnt n~ ~u~h~nt~ o
~t~n~ard~. ~Axol ~c~-.rity~ on~ d ~y o~o~1n~
th~t oxtrac~ v~ llty t4 ~tabllls~ ~iarotubul~s
:~ ln th~ 0~4 ~annor ~a ~ ntl~ taxol ~FIC. ~). rh~ thlr~
l~ne Or ~Yidsnae t~a~ tAxo~ on~Aln~ in ~ o~ll
wa~ 1~y puri~yln~ ~xol on NP~ ~nd lden~leyln~ tAxol
~1~ purl~l~d ~Ampl~ b~ nucl~r mA~nnt~ o ra~nan~
3S ~n~ly~Lo. 4erlYa~v~ o t-~xol hnd ~ddit1~n~1 pr~ r
~rs alBO ~nt~ied by ~PI,C ~n ~ach 1~ c~ll lin~. ThiE~
wa~ aoaampllohed hy ex~ract1ng ~ o~ ~h~ a~ n~,
2~9.~22
a3 -
runnln~ ~e ~xtrac~e on ~ 1~, e411~ctlng ~or ~Q~k~ ~nd
t~ting the~ aa~ ln ln vitro ~l~o~llbul~ ~tA~ in~
a~ya~ a~rlal~ and M~thod~).
Th~ lo~ o~ tax~l pr~du~ltd ln un~lial~d ~ul~ur~l-
S o~ m~dlfa av. ~lck~1 C~ll lin~ CStl, ~3, ~, nn~
~3 Taxol l~v018 w~re ~-3 t~Q0~ h~ U~t~r ~h~n t~ la
Po~nd ln ~x.t~ x ~r~ yoUr~ ~te~ ,o ~ sr~lnod by ~P~C
an~ly~1~. Ev~n ~nor~ intG~r~t~ng wer~ th~ IIPLC pro~ 3
roYeal~ng ~reiv~ou~ly uni~nl~i~ a~ with 'c~ ke
~ct~vi~:y, and ~ro~ dl~r'~renGo~ ~ong o~ n~. For
6~xa~bpl~, dif~rQn~a in ~ N~C ~hromta~r~ pro~ o~
oRll li~ SF~ nd 9-3 ~ ~aren~ wh~n ~cm~r~a wlth
thel ~IPI,C pro~ Or c:~ll llne~ CR~ hown ln ~. C,
und~x condl~ion~ AID d~in~d fDr ~IC. 6. P~k 4, ~
whl~ t~ taxol-lilco actl~ y sn~ wh~n 1~ ~ra~nt
~n C~ dl3: lni~h~d o~ ~,b~t ln 3-3 . P~c 5 1
p~e0~n~ in CR-1 but dl~lnluh~a in ~-1. Inter~tingly,
~lth~x p~kB ~ nor S ~e~unt!l ~n C~-l w3~ d~t~C~d ~n dlrQc~
~xt~a~to 6~ d~ lok~ undar th~ ot)n~ on~ ~od
~o d~ t th~ ~3~ o~ o~llu~ ~d ~ul~ur~ XtPaclt~.
It ~ tpest~d tllat oths~r pr~lt ~ wlll r~ult i~
dl~r~nt ~row~h p~raD~et~r~ o~ aul~ arl~ uGad ~r
d$rrer~nt HPLC m~thO~!IB ~ ~11wa. The ~th~
illu3tr~t~d ~or CR'~, ln FIG. 6 ~r~ de~cxlb~d 3~ex~sln.
O~h~ mothod~ ed ~n hydroph~bialty or o~h~
~l~tlr,oul~hin~ m~ d~ for ring~contQ~nlnq ~tru~ture~ ~r~
~Ult~
~ho BU~pen~lon cult~r~ l~no~ ~3 ~n~ 61~1 ~r~ grown ~n
lL ~la~Xs ln Ga~obor~ ' o ~B ~e~a 1~ V), ~lot~ c~
uto~ Yud tunçl~l G~ll wall ~a~p~nl~ nd 13p~X~4) ~nd
~bl~lc ~ltls 02 h~ n~t~ nd
~5 m~roury) ~llcitatlon ~r~ ~ult~bl~ llcl~c,r~. Doth a~l
11na~ a1Loltation ~ may ~ult ln ~1Dtt~t~d
1~Y~1~ c t~xol, 1~ prQ~ or~ ~nd t~xo1 dloriv~lv0~.
,
,
...... -- . .. .. _ ., v ~ v ~ V ~ v l .7 '`: ~ ' J I S v ~/ 4 ~ O
2~69~22
D~L~ 7
Indua~on ,~ tu~
a~ ~ald
In or~e~ ~o lnd~uoo mut8~tl~na whioh ~oducs high 1~
o~ t~xol, 1~ oo~xn~r~ an~ ao~lv~ tivo~ o~ t~xol
1n ~ l linQ~ CR1, s~, ~Rl an.d ~:3 Ar~ o~d ~o UV
~xr~dlRt10n ~r~a~nt ~nd ~a~1 mu~qa~n# 5uo~ ao ~S.
Bsl~ction ar~ t~rlA and ~e~o~ o2 ~uleur1ny tax~l~
l?ro~ucin~ c~ll line~ cordlng to ~o~e ~ o~l7 Wlf.
hQ~in. In th~ shlon, ~.Ut~t~Oh~ hAt ~sor~ h~
outpUl~ ~ the ~xol~p~hâuclhg ~ , m~y b~ t~d.
~e~. ~n
A n~vQl ~D~thod o~ c~crag~ pr~rred ~or ~ua cu~ r~-~
1~ ~a~ tc~ r~fluw lnot~at~on ~ p~rAturo ~o 1~-15'C!. ~:row~:~
Wl~ ~low~ to a hlb~rnat~on-l~k~ ~t~to. To re~urr, c~all~
tO norm~ t~mpQr~tursl WA~ ~lowly ral~-d to tho
opti~ ~or g~o~th.
llu~ a~ n~ CRl ~nd ~G3 arc m~int~ln~ on
~ r~n~ ~ar~6 plac~d ~nsid~ NAg~n~a ~A-7 v~sssl~, while
shaX~r aultus~a o~ ~3 ~nd S~ ar~ Aint~ln~d ~n lL ~ha~r
cul~ur~ k~ and Bu~cu~ 3d ~ery 2 W~
Cry~r~rV~lon ~or lons! ta~ ht~r~g~ o~ ~11 cult~e~
CRl, ~:3 ~ nd ~ urlng the ~ ?h~ of ~r~ Wa~
a~com~llshod by ~lng ~t~nda~d ~yopro~0r~a~ion ~thod~
U8 ~ ng ~orbitol ~nd D~80 ~ th& ~ry~prot~t~nt
Cr~opr~ v~tlbt~ wa0 ~ho~n ~o hav~ ~r~ t~ntia~ ~o~
lon~ tor~ pros-~rV~ Qo of coll D~llt~rss Yho~ b~o~yn~h~tlc
cP~olty tor 3ynthQ~i~ an~ ~ooumu~ n of ~ond~
mot~bolit~ romn~2~ inthct ~o~lowing r~l~o~ r~m ~stc~r80e,
1'h~ growth o~ ~UB~n~lOn C:9~ 1 lin~ ~1 in ~irllt,
35 ~t~rr~e an9 p~rIusion bl~r~ot~r6 ~ult~ ln ~xol
~roduot$~n, Alt~rnat~Y~l~, naw r~4C~3r~s mAy 1~ d~sLqn~.
.. .. . . . . . .
- : : . . . .
, , .
- , .
.~ , . . , ~,,
, - . :,.: ~. .. .
~ ' ~Y~.: I ~Y~ V ~ I . ~ 1 . ' J _ I V_ ~ V J V V J~ <.)
~0~91 22
- 25
~hQ p2:a~0r ~d~ t~n~ o~ ~nt~lr~n~ont4~1 condltlc~ s~oh ~
~a~ po~ltlon, ~l~eolv~ o~s~n, rat~ par~inq ~nd
a~ee~ gita~lon, t~m~er~tllr~ o~ tho grbWt~ ~dlu~,
~utri-nt~ o~7an~c Elup~l o~nt~, r~ov~1 o~ orld~ry
~ro~ua~ ~rom th~ diu~ ~rbugh ~h~ UB~ of rQ~ln~ ~nà
~licltation ~r~ r~ul~ t~ ~ptlmiaQ t:he ~y~t~m0.
~h~ growth ~ the c~llu~ lln~ uit~blo ln a
"mem~r~n~ flo~ lor~ o~. ~hi~ ~y~to~ h~a nlltriQr~t
~olu~lon rlowln~ o~r tn~ br~ ~hi~h 6~p~rt;~ tho
c~llu~ ~nd 1~ ~P~x~ulated. Nutri~ntf~ aan b6~ d. ni-
ne~dsd ~y ~eR~u~in~ conductl~fity nnd th8 pr~genc~
t~xol, ~axol der~ tiv~ an~ pr~aur~or0 ln thQ meA~u~ ~an
ba a~ y~d b~ ~a~pl~n~ ~ra~ ~ho 301~tl~ In llna. ~hl~
~t~ Up By~t9m 18 analogou~ t~ thR g~o~rth o~ thR oallu~
~5 cnll 11n¢ CRl on m~ r~n~p x~.~t~. '~$8 ~yG~ an alç~o b-
~ 12~ ~y hav~ng th~ nut~l~nt ~ ray~a o~to th~
o~llu4 Int~r~ltt~ntly th~ou~ a ~yat~ oon~$.t~r~g ~ ~
n~ , a p~rls~Altic ~u~p ~nd ~ r, thor~by ~u~plying
~h~ nut~i~n~ ~o~ unl~Dr~ly ~o th~ en~ nllu~ nd
2~ o by lncrua~ing nutr~nk u~ak~, ef~ial~ncy, ~n~
mlnl~nls5,ng nutrl4nt U8~-
~lle th~ in~t~ntlon i~ ~uu~;~p~ to v~iou~
r~odi~14ati3~s ~ alt~ tl~ ~orm~ o~lc ~dim~n~
thor~ v~ boan hown ~y w~y o~ oxnm~ n 'ch~ draw~ng~
~n~ her~in ~Ar~ da-cr1~a ln d4~11. It ~holl1d ~D
undor~toa~, how~or, th~t lt 1~ not 1nk~nded to l~mlt ~h~
1n~ ~ntion ~o tha ~artia~ r ~orm~ dlac1c~d, ~u~ an th~
aon~rhry, t~ tan~1on i~ to c~o~0~ ~11 nodi~aJ~io~
~o ~e~ulv~l~h~, as~ al~rn~t~v~ llln~J within ~ lt
~nd ~COPB of the inv~ntion ~ ~et~1n~ by ~h~ ap~ndod
~llaim3 .
.
- ' . .;' . '' ' ' "
.
.~ :- . . " , , :.
- : , , . " ,
206g:122
-- 26 ~
~I~I Co~p~rl~n ~f ~x~ v~ und ln dl~r0nt
E~larlt tl~u~ of th~ g~n~ TavrU~ nr~ ~xpr~ed I~D
p~roont~g~ o~ e ~ry w~i~h~.
~--,_ =~_ ~ e~ ~_
~ b~ ~ ~ ~ ~
~ep~udon~ ~ h~h~ ~
~ . _.= ,. ~__ ___ _ ... ,._ .. .. ...
5 ~_ ~ , ~.01~ __ _ --_
tnnn~ 0.001' 0.0~1~ 0.~ ~ ~ 3~ û.~
O.ODl`O.~OIr 0.û0S3'
i~hlro O.OW D.~33' O.~ID7~ 0.~117
~ __ ___ ___ .._ _~, __ ..... . ~,~., __
0.003' 0,~61 ~ O,QD~ 0~002' 0.00~-
.003" O.~ 0.01~4' 0.0141 0.00~
l),0~15' ~.0(~' O.OlS6'
L ~_.. ~ ~ __ _~ _ _ I
~Wl~erup~al., 1~9U~Withuup,K.M.,S.A.Lo~k,M.W. S~s~D,T.
nd ~.M. ~lu~chil~ pp. n~dle~ ~ount~ of t~l comp~blc t~
tho ba~k of 1~ ~ lia; Andy~is and l3~lnd~nl J. Natu~l Pr~dllct~ ~3:1~4~-
l~S (1~90).
Vidense~ C~ Rl., l9~0 ~Yide~ t, N., Y. Lim, A. C~np~li and C. ~ nn.
~5 T~xol eon~4nt ;n b~rk, wood, xoot, Icaf, twiSs ~d ~dline fr~ everal tsxus
Ip~c~. J. Nat. I'rod. ~3~ 1610 p99~.
Wlc~nl~sinhe ~nd Art~
.. ..
.' ! , !,
'. . . ' ~ '' ',
2~69122
-- 2~ ~
T~ II Com,~)o~itiDn ~ Gambo~ B5 n~odiuDt.
~al ~al~
rn~lL
Ammon~um ~ulfat~ 134.~0
B~rlc acid 3.000
Calcium chl~d~ ~nhydrou~ ~13.2
Cobalt chlo2ide h~Ahydr~te 0~
Cupnc 9ulf~ ~n~hyd~t~ O.OQS
D~s~iu~ ~TA dihydra~ 37.250
1~ Polrous slltf~te hcp~ydr~t~ 27.8~0
M~n~u~ ~f~ anhydsous l22.0~0
~nego sulf~te monohyd~to 10.0Pot~sa~uln iodid~
Pot~si~n nit~ ~500.0aD
~odtum molybdate dlhydr~t~ 0.2~0
~odlum phosph~te monobasi~ ~nhydrou~ 13V.gOO
Zino sulf~ heptahyd
V~
My~lno~ilol lW.~Thiam~ C1 10.0
Nicotlnlc ~ 1.0Sug~r~
a5 SuC~n~e 20,0~.0
,
- . '
.. '
. .
2~69122
- ~B -
M~ifl~ nborg~ B~ m~un) I~Dr g~owth of c~llU
n~al S~}~
~L
Ammor~um ~ulf~te 134.00D
s sario ~d 3-000
Cdciurr chlori~e anhy~ .24
Cobd1 chlo~ide h~hydra~ ~.025
Cupna s~lfalcpent~hyds~tc ~.OQ5
Pl~di~ TA dlhydr~t4 37.2~0
~o~rou~ ~ul~t~ h~ydrate 2?.~50
b~n~alum sul~ate an}syd~ou~ 122.0gO
M~galte3~ sulfat~ mon~hydrata 1~.~0
Pot~u~ iodids 0,7SO
Potas~um n~atg 2S~.O~
~ium molybdah dihydr~te O.~Q
Sodium pho~ R~e rns~ob~sie a~hydrous 130~5
- ~inc ~ulhto h~p~hytlr~to ~.(~OO
Vit
l~y~inosltol 1~.~
2 0 ~amina HCI lû.O
Pyrldoxine l.O
NiGo~nle ~cid 1.O
Sug~ls
Suc~ose 2O,~.O
a~ Gluco~e 2,~
P~ucoD~e 2,S~O.O
C:~ln hyds~ly~atc 1,O~O.~
.-
', , `, .,: ~ !
~,
' ' ~ '': . ,:
..
2069122
2g --
~A~I~ IV M~dified ~ r~ BS modil~ fo~ rth of c~ll suspens~ons.
B~al S~lt~
Arnmonium sulfat~ 134Q~O
Bo~ aGsd 3.aOD
C~ob~lt chlonde hexahydrat~ o.ms
C~o Wlgdte pe~ yd~t~ O~i
dium ~T~ ~3;hyd~ata 37~2SO
P~rmus s~ oh~ptahydrAte 27,~54
1~ ~nesium sulfate allhy~nut 1~2
Mitn~nc9e g~llhte n~nohyd~dte 10.~
Pota~um ~ e 0.~50
Pot~s~ium ni~t~
Sod~um molybd~ta dlhy~ate O~Q
Sodiuln photphatc ~nonoba~ic dnhy~ous 13U:500
~:inc ~ te heptahydrato
~tr~
~y~ino~ltol 200.0
llùan~ino EICI ~o,o
2 0 Pyridoxln~
NicoU~lc acld ~.
~u~a~
Saoroso 20,~0.0
2,500.0
~uc~s~ 2,50~.0
ein hydtoly~s~e 200~0
TAll~ V
_____ ~ _
(~JI lu~ ublin~ (d4ys)Ta~ol ~ _~
CR~l 1~ O.OOl~o
~. . ~_ _
3~) ~R-l 5 g S~.001~.016
- .. ... _ ,,, _ . . ~ ~
3 3-~ 0~1-0~08
~ , . _ I
LG-3 15-1~ O~Qol^O.OQ0
...... ~ _ .. _ ~
LCS-3 1~-16 0.001~0,~1~
~,~,,, . ,,", ~_ __
.
~0~22
- ~o -
~ha ~iere~tubul~ 4t~hll~1n~ bio~ y i~ 6ed ~o ucr~n
~or ~ b~ 1~ ty of ~ lti~ to ~tubllle~ tho
~or~tlon c,~ ~nlcrotubu~4~. ~h~ra 1~ a dllr~t, ~rs~aloti~
~oxrelAt~on b~we~n ro~ul~3 11~ th~ m~c~ubu~e lDI~ ay
and th~ ch~ rapa-lt~ ~Ct~on o~ ~ ~om~?o~ n in o~na~r
l a~ay~ ~h~r~ror~; ~ho ~ro~ubul~ n~lliz~ng
Oa~ Ay i3 xeoognl~ed ~y tho~ o~ ~Cill in ~;h6~ ~r~ ~ a
OC~ n~nl;~ tel~t ~02' ~ aCtiVQ ~he~c~th~r~aUtlC ~g~nto ~or
r~ ical ~rl~ln.
~o ~ndu~'t th~ ~lo~ y, ~uxon~ rottlbul~
~rot,~in~ are pr~ od Qooord~ ng ~o ~o m~od~ 0
lS ah~l~nski ~t al . ~ lg73 ~ u~ r~h cal~ br~in~ .
Endo~nou~ m~rotu~ule-a~iso~at~d ~rot:eln~ r~
ramo~r2d ~ro~ prot~in~ ~h~h h~ ba~n ~yclod on~ ti~e,
~y oyclln~ ~ tlme~ lr3 et-~ ~>r~a~o~ o~ luts.~o (~l~m~l
l~nd Lln, l~a~). A~t3r co~ tlon o~ tl~ a~ , th~ t
2~ t~bul~n pcll~ obt~n~d i~ ~olubil~ ln o~ld ~ bu~r
~nd 1~ ~uxth~r ~ur~fl~d byphq~ho~ellulosG ahro~*og~n~hy
acaordin~ ~ W~ngart~n ~ al. tl~75).
~o~ay u~;in~ ~a~ iol~ ~lcro~aopy 1~ p~onn~ d~
~olloot~:
~1~ tho t~t ~x~ract~ ar~ tho~ ~olub111~d ln P~
burP~r a~ crl~e~ ~b~
uffor: 50 m~ 4-plp~r~ 4dl~hAne~ul~ania a~
~Pi~) a~ l?lI 61 9; 1 ~M ~n~no~ium ~ul~ a
~thylene glyaol-hi~ ~b-a~no~thyl 6~hçlr~-N,N,N~ ,N~-
t~tr~acetlc h~ld ~ECTA] tu~ulln and ~ P ~toak.
2 . ~ ~
. a ~nd
3)
~pp~a~i~nntely 90S ~ l'a2~us at~m ~zpl~nt~ ahow~d
3S cnllu~ ~n~u~tlon ~t~r ~ouæ 2-4 ~/~aknl o~ irll~int~o~.
w col~red CA~ we~fd BE110Ct:~d ~ A~IIt~d erOl~l th~
~xpl~nt, nnd ~ubaulturad. ~ UB oh~ctori~ticall~ ~Ow
- . .
- , '- : ~ ' ' .. :
L. ~YUI I ~Y~ U ~ 1 . J
7L ~ vrlr I V~L l~ Jl~n ~vr~ n~ 4 i
2Q69122
- 31
~n~tlal ~lov growtn, ~nly doubl~nq ln ~1~out 3-~ ~onth~ o~
lon~er. ~th~r dl~ ulty to be ov~room~ 'ch~t ~o~t~
sallw~ turn 2~ ~row~ aolor w~in 1-3 w-~ko ~f~ex
~uhoul~uru!, ~nd ev~n~u~lly d~. R~d-~Olor~A ~xudat~ w~r~
l!lno~Qr ~lqn of lm~ n~ ~11 d~
Cl~mp~ o~ y~ aolo~Qd~ ~ri~bl~ a~llu~ w~r~ ~3al~ed
f~r ~u~c~llturln9~ d ~nd ~rown colar~d c1~p3 w~ro ~O~
at~d. S~ tabl~ olum~ were o~n ~urro~d~d wlth
brown-aol~ d ~dsad) c~ .u~ c~ , tho llv~ c~ t~o~
ap~r~ng ~ nodul~e ~ro~ud~nç~ rrom a br~ colc~d ~s
ol;' callus.
3 ~ 1 ~ ~Y~
~rled ff~thyl~nQ chl~r~ xtr~ usr~ ~isctolYod in
~th~nol ~or HPL~ ~n~ly~ n~n~Atl~ oon~ t~d ~
~ w~Pr~ 6K ~ver~uid ~hrb~Q~o~r~h
ln~eator, Nat~o ~DE n~ultl~lv~nt d~liv~y ~y~t~ nd
s~a~Qr~: 4~4 tun~bl~a Ab~Or~n~ d~ta~tor. A ~yna~x-~o
n pheny~ a~ 4.~ mm x ~0 m~) wlt21 ~ g~r~ m~dl~le
w~ UDoCI~ ~ha ~ob~la ~nahe~ aon~ko~ Or ~ ~thanol,
~moni~m ~q~ta~ bu~r ~n~ ~c~tonli:rll~ ~un ls~or~tic~lly
~4t 2~:41:39, ~beor~no~ w~ da~cted ~t th~ ~b~orp~l~n
m2~xlmum 0~ t~XC~l ~hl~ 227 ~. Auth~ntiC ~tAn~rd~ o~
t~xol (1~, c~ nnin~ ~a) ~ ba~fl~n ~3) w~ro u~à
to ld~t~y the p~kd ~a- 6) ~ t~ndoxd~,
2S ~qht: M6thyl~ne chl4~ xtraot ~ro~ C~-l. Poo)~ nu~r
4 ~nd 5 ar~ us~ld~nti~ chibltln~ ivl~y in th~
3ior~tub~ bl~ y.
,: , , ~ , ,
.~ ' , .
. . .- . . . : , ~
~ ~ ,
V ~ I V r ~ I f ~ J ~ ~. J ~ G . ~ ~ V
2~69122
- 3~ ~
.,
`:
x,~g~r~oo,~
Irh~ r~f~PArg~n~ t,~d bslow ~r0 in~or~ r~ln
by ,r~rbn~ ~ ~o ths ext~n~ t~A~ th~y ~uppl~ nt, ~ n~
pP ov i a~A a ba ~ ,und i o~, v A~ ~A~ch ~hod 4 ~ y,
3 t~,ohnlq~lss,, nd/or c~m~osltlon,3 ~pl~yad. heLAeln,
~rodollus, P. -~ B ~ot~nti~ loe of l~noblll~sR~ion ln
pl~nt a~ll bi~t~hnol~ frr~nd~ 10~4uhnolagy, 3s~o-
~5 (lg~S)
,~odoliu~, P. I~mo~ Ad P1~ t; coll~ oura~
1~ bioch~lod~ n: ~ior~aactor l~obl~ d ~n,s~ ~nd
c~ d: M. Mo~-Youn~ vi~r A~ d ~ci~n~ ndon
~nd N~w York, ~Ba, ~p. 167-lg6.
chrls~n ~t ~1., Prndt~tl~n o- ~x~l or T~xol-liko
Co~po~lnd~ ln asll CUltllre~. U.~, P4~ant No. ~,0~,304
~g91).
~ni~ N., A.~. Gr~n, D. ~u~n4~d, F.
Gueritto-Vo-sloluin, ~. NAnqat~l and P. Potl~r. A hlg~
e~loi~nt, practical ~pxcach ~ n~urnl t~xol~ ~0 e~
Amu~. Che~. ~oc~ 110; 5~17-5~
Dor~ohowar, ~2~C~ E.X. Rc~n~ky~ roo~ow, ~.~.
L~nqn~atce~ ~nd D . ~ ~t~ qer, ~h~ I tr ~ al ~ t~xc~l ln
patlsnt~ Wl~n ~dvanc~ ~nna~r. ~ano~r ~rt. R~ort~
7~ ) tll71~1i77 ~1987) .
F~lzone, C,P., B~n~i, A.J. ~ Le~mt~ J.T.J. ~l~g2).
2S ~h~r~ct~ri~ on o~ eaxol ln m~tnyl~n~ ehlori~ by N~
~ec~ro~oopy, '~otrahsdron ~4~t~ro 3~ 2.
~ul, H., M. ~ nd ~. T~batU. ~nduation o~ ~h$konln
by ~ndcg~nou~ poly~acah~r~ d~ ln
~ryt~orhl~n o~ u~n~lon oult~rea . PlAnt C~ cpoFt~
73-76 ~1990).
Gamb~rg, O~h., 11.~ r ~nd ~t. O~l~a. Nutr~nt
requlr~m~nt~ o~ ~u~p~n~on cultur~ oyb2~n root c~
E~p . C~ ., 50 ~ g0 1 1~68~ ~
~u--~e~-vb~gbl~n~ c~ Lhl41~ ~n~ ~. Po~r. TAXO1
And d~ri~Ativ~s: ~ b~oq4n~t~ th~ . Jourr~al o~
N~tu~nl P~oducts, 50 ~ 9-18 (19a7) O
H~al, E. ~n~ C.M, I.ln. aluth~kat~lhd~d ~ol~k~rl~tian
o~ tu~ulin: oh~r~tQrl~tlo~ o~ th~ r~a~t~on an~
~pplic:~ti~n to t~a lnr~o~aal~ pur~tl~t~on ~ ~ub~llln.
A~ch. ~l~c~e~. Bio~hys., 20~:29-~0 (1981).
~a~n~in, P . F . E~t~lrl~ appxoa~ to th~ l!on~tlon o~
Qcond~lry nstu~l prod~t~ In pl~nt ~oll ~u~p~r~aion
J'. Na~, ~ro~. 4~ 19~$).
,
:
..
L ~ Irl~ 11 VLL IC~L~ L~ n JVr~ n' ~14~ C~
~069122
-- 33 --
Hor~ t~, S.~., L. Lot~t~i~, J.~. Nanfr~dl, W. I~loll~do,
J, ~i~rneB~ ~.N. Roy, ~.~. 6ahi~ L. ~orb~ro. ~n~ R.
~h~. Irax~ls MeQhanl~m ~ ~c~lor~ ~nd r~ t~t-o~ Annal~
N~ York A~do~ o~ ~oi~ e~, N~w Yo~c; Th~ d~y, v~l.
466, Pp. 7~3-743 ~19
Ha~ , N~ d 6 . N . ~1~aDh~ 1nk4s~ b~t~
e f~t~ o~ t~xo:l, oc~lohlcin~ ~nd ~TP on tubulin
~olym~ri~t~on. J. 0~ bi~lo~1chl ~haml~ery,
2~ ~3) S 1~2-134~ g) .
1~ Huan~, C.~Ø, ~.G.I. ~nq~t~n, N.~. ~aqri ~nd a.
r~n~ya~e. ~w t~xanes :ero~ vlfoll~, 2. J.
o~ Natural Pr~d.uctt3 49 t~ 19~S~,
Xar~oh, R. arld ~. Mitton~or~er. Ultravlol~t 3:ad~tlon
~ ncr~ nl~ot~ne pro~uc~lon ln ~1~ ~allus
o~lturo~. J. Plant Physlol, 3~ 0~ 1990).
Klnq~on, ~ . N~wkln~ ~n~ Ov~ngton. Now
~x~n~ rrom T~u~ br~ . J. Na~ur~l Product~,
4~ 66-470
~lcGu~r~, W,P" E.K. P~win~Xy, ~.~. Ro~n~h~in, F.a.
cr~tm~ln~, D.A. Et~ingor, D.~ t~os 0, P~.cù ~c)nohQw~r.
TAXO1: A un~ antln~ls~tlo ag4n~ wl'eh ~lgnlf t
~ct~vity ln ~d~ncad ov~rlan epl~ al n~o~la~. Annal~
o~ In~rnal ~S~dlcln~ s~?3-~7g tl95~-
McI.au~3hl$n, J.~ . Hill~r R.C. Pow~ll and C.R. a~lth
Jr. ~9-Xy~lroxy~c7a~tln I~ 0-~oac~tylo~ph~l~nnin~,
and l:O-d~na~tyl~axQl ~ N~w ~n'clt~2~0r tax~ne~ ~r~m T4
~lllch~an~. J ~ Na~ur~l P~adut~ 44t~ 12-31~
Mlller, R.W., ~.G. Pow~ll an~ C.~. 8~ h ~r~ Antile~omlc
a~knl~d~ fro~ ~U~L~o w~ chl~n~ Zuao. g. Or~niQ
Ch~n~try 46:146g-14~ 81) .
w~, }S., M. Il~y~-hl an~ ak~y~ cou~u~ation o3!
antlnea~la~tio aC~Qnts by ~ nt ti~l-U~ ~ulturo~, n~
Prlm~ry ~n~ ~eoond~y M~tabol~m o~ Pl~t ~11 Cul~ur~;
~!d . K . N . Neum~nn, w, ~ xd . 8pr ~ n~
3s ~erlln ~ lb~rq 19~S, ~ol 3~, pp ~3~-a~6.
,~lqo, T. and F. ~o~. A r~vi~ me~ or rapl~
growt~ dn~ ~iOAI~S~ wlth tob~ccc~ t~Aua ~ultur~s.
Phy~lol Plan~. 15:473-497 ~1g6~).
P~rnOo~ n~ . Horow~tz. TeXal binq- to poly~ lz~d
tu~ulin ~n vltro. ~ ~tll sis~ t. l) ~47s~4a~
.~ ~19Bl).
Payne, ~.~., Shuler 2~.~. and P.~ llu~ lg~73 . L~rge
90~1~ Pl~n~ ~ell Cultur~. ~n~ L~r~ Baale c~ll Cu~tur~
T~chnolo~y, Ed: ~orn R. ~der~on. l~ne~r Pu~ h~
4~ ~unlch, ~ snno., ~4w Yorlc. p~. 194-~9,
: . .
. n~ n,~ n ~ .~ L I J~ r~ uLc ~ nl~1~Cr~ 4 ~ 1 0
2~6912~
-- 34 --
sohl~I', P~B And ~. H~rbW~, T~xol ~ bll~
nllcrotu~ul~a in ~ou~ ~lb~obl~t a~ . Proo. ~ln~, Ac~d.
8~1. 77 ~3) ~ 15~l-15~s ~1~89~ .
8h~1~n~Xl, M., ~ !cln dnd ~ n~r. M~o~tu~?Ul~
~e~bly ln ~ sl~en~e o~ add~d nuol~c~tlde~. Pr~c. PJa~.
Ac~d. 8ai, U~A, 7~ 5-768 ~1973~
~lCl~rod, O. A~ kj~X-~r~eX, Rlgln~te ~o
lmmoblllzation m~trix 1~or a~ . Tr~n~ ln ~lo~ohnolo~y~
~71-78.
1~) SnAd~r, R. I:tv~rvlQt1 o~ ~up~ly ~u~o. ~o~k~hop on ~xol
~nd r~ Cu~ror~t ~nd ~-~tu~ tl~ p~n~or~ad byl
D~vel~ nt~ 4r~eutl~ gr~, bt~ n o~ nc~r
trhstr~Qn~, N~tl~n~ nc~r in~tl~ut~ 90). ~traat.
80u~htan, ~.W. and ~. Bu~lngh~s. D~ctto~ry o~
1~ A1~A101d~ Ch~PmAn ~ H~11, No~ Y~rX ~193~), pp ~o~, 1024.
~u~n~ M. ~nd ~o~dall G.A. (19~5) . ~n'c~tumor nlk~lol~,
nl Alkal~ ol . XXY. pp 6-18.
~u~Pne~ 19gO). l~ o~ nd ~arly dbv~l~p~ent o~
~ol. ~tork~hop On T~xol And T~XUSDS ~rront and ~u~u~
2~ Pars~o~t~v~. Nd~iOn41 C~na~ Instltut~ 0.
~b~tract.
Etu~n~so, ~. ar~d C.~, Co~d~ r A~ old~
~k~ id~/ od. A. Bro~i, Acad~ Pr~, Ino. ~BS),
Yol . ~nr. ~p 6~ . 0 at~d S7 ~g .
~u~no~ . Di~aov~ry ~n~ y ~ lopm~nt c~ ~xol.
Nor~coAop ~n t~xo~ an~ ~xw: Currunt ~n~ future
p~r-~-ati~a~. Etpan-~n~e~ b~ ev~ ntal 'ch~rAp~ c
pro~rA~, 3i~ on ~ n~r t~ nt, N~t ion~l ~a~lo~r
ln~t~uto ~1990). ~ o~.
~hlg~n~ n~n~ t Al. ~ Ph3~a I~ trl~
~r taxol ~n ~aoond-l~n~ th~ea~y ~ o~arl~ rcln~ A
gynscoloqlo on~logy gro~p l-tudy~ P~oo. 1~. ~oo. Clin.
Onaol~ 9: 156 ~l~P0~.
Thr~ nd I.PI. ~hlt~h~ bts~ n~
~3 to in~uo4 th~ ~or~tlon o~ ~oonda~y pr~un~ in ~lQnt
ti~us o~llturo, In; b~n~pul~tlns~ on~l~xy M~tAbe7~ m ln
Culturl~, Bd.; R.J. ~obin~ ~nd M.J. ::. Rho~o. ~Prldy~
~JniYe~-lty Y~s~, Ca~rlag~ 19~B, pp. ~ t.
vl~i~n~X, N., 1?. Li~n, A ~amp~ll ~nd ~:!, C~rl~on. ~X~51
aon~nt in bar~c, wood, root, leai~, ~wl~ ~nd ~ ling ~ro~
~eYer~ oles. J. ~t~ P~o~. ~3:~60~-16~0 (l~o).
~nl, ~.C., H.L. ~rl~y~ C. Wall, P. Co5~qon an~ A.~.
~toRh~ll. Pl~r~t antltumor ag~nt~ ~11. The l~ola~:ion ~n~
~t:rua'cur~ o~ T~XOl~ a noYDl ~ntll0uk~mlG an~ titwllor
.
,
. . ~ J ' .~ ~ r ,11 . V L ~ V 1~ 1 4 ~ j 4 ~ 5 ~ D ~ d
2 ~ 2 2
-- 35 --
ag~n~ ~rO~ T~XUB b~vlr~ . J. ~herlo~h ~h~lCQl ~oc~e~ty
93 ~ 23~5-~327 ~1~71) .
Wf~athoro, P.J. ~ e~ X.h. ~1~8~). Ra~n~n~ion
pl~nt~ lng n~rlen~ aultur~. In v~tro ~llu~r
D~ lo~ ntal :~1olo~y. 241 ~7-73~.
wolnçlarten, M.D., A.H. L~ek~oo~ 8.-Y~ ~o and PI.~.
glr~hn~x, A prot~in fa~tor ea$ent~al ~or ~lcrvtubul~
~ a~ly~ ProC. NAt~ Acad. ~oi. U~A, 72~18g~ h2 ~197~)~
W~ ru.p, X~., 8.~ X~ t~sko, T~ lo~zi ~n~l
a.~6t MIl~Ch~ nxl~ Bpp. n~ oo~taln a~un~l of t~Yol
COmP~b1~ t~ th~ b~ark ot' 3'~1SB ~r~vlr~ a: analy~ an~
i~ 1On. J. N~tUra1 PrOdU~tB ~3:1~4~-1a~S t1~~-
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