, ( Rifeve fo Thea '4/~ Gee '¥ 2 ) f , ~ hither S TY me prepared by J, Lederberg Biol. Labs.,Columbia Univ. COLCHICINE 1. Introduction oes Chemistry cH Pharmacology cwioch 2. The C-Mitosis Ry CHs Plant cytology . | Animal cytology He 3. The Physiology of the Ciiitosis Similarities to anaesthesia Physical etfects Bio-chemicaL eifiects 4, Aliium cepa: the colchicine susceptibility gradient o. General Considerations 6. Bibliograpny il. Colehicine i8 an alkaloid present in corms and roots of various Liliaceae, particulariy Colchicum autuwnnaie, the meadow satiron.(Kiein & Pollaut '29) Although it has never been fully synthesized, the researches of Windaus and others indicate the formula reproaucea here: (from Henry % '39) It will be observed that the nitrogen is on a side chain and tnat Ve wo! 1, ~ there is an acetanilide nucieus present. (Note 1) tuluiy 2 anobetty gator vader Saffron extracts nave been used since Byzantine times as an empiricd specific for gout,(Rushy et al.,'30) Water anc uric acid excretion are stimusated. However oom infiamuetion and respiratory depression rrequently oceur so that this drug is not widely useu in modern practice, temperature plays an important part in determining the toxicity of this drug, warm biooded animals, or trogs Kept at a high temperature ,naving 400 times wie sensitivity as cold-biooaed.(FPunner,...,'33) Jacobson ('25) reports a specific paralysis ot caraiac parasympatnetic terminations. 7 me dus te tre apia protic > yreig Soe He! Lortniori finds a moditisation of the Arneth Leucocyte count, a Lowered Tenal thresnola tor glucose ana a aecrease of iiver glycogen, ail intdica- ting an interference with renal mechanisms, Clare &@ Barnes '40 fina that colchicine poisoning, iodoacetate poisoning, and adrenai-sortical deti- ciency are ail relieved by the administration of salt-water or of cortical | extract. co haly| there are also scattered reports in the French Litgerature of stimulatory etfects on parathyroid and testicular tissue. It is piain that the general pharmacological e:ieets of this arug have not as yet been reconciled in any comprehensive theory on the mecharism ot its action! Be It is not on the basis of the above-listed properties that Golehicine merits the attention that it receives from biologists touay. A number ot other e.tects of this arug have tren recorded which are tfaceable to its etiects on t pdf Septet cone uate ae ceil-division. The idetions, described below, whieh Colchicine introduces iato celi-division nave been caliea the C-Mitosis (Levan '3s). The early nis- <¥arrttin, $ poie tory or its elarifieation has teen reviewed qwuite aaeyguately by Dermen '40, um _ a . bg oe Fyte ‘sy, anu wili not be repeatea here. The work or Levan ('58,'sy) aescrites the cemitosis as roliows. he-metertat- ig tne root-meristex, o1 Aisium cepa; te concentrations of the arug varying rrom ,007bo to 1, applyea ty merely grow- ing the roots in the test solution: Prophases proceed normaly, and , polar aps may appear in the earliest. CAAigwred ort Stages. TM spindle however aoeS not become organizeu. AS a result the wetapease Aan & dings ws never oriented, azd, the chromosomes do not appear on the equatorial plane, celi Insveaa they remain scattered about the ,apparently Ior as Long as seven or eight hours. ne Kinetochore is aiso somewhat iniLuencea for it does not ai- vide — iw wou thea in oa normai rhythm. The sength-of-tire chromosome does however soon spiit, ‘e2d-—repuision orsome Other. 1Opae spreads the halve out atad Cond so that x shaped chromosomes appear, the point of juncture being the nsptit tad Chuprnsrmied Asset zine FoGhors « vent tnois too aiviaes syielaing perre in a tetraploid sasera 4 Gar. * ee agkalan TeLo hasic ent ne vion now ensues, cut the uilscorpaniesetion of tne cnromu- somes resuits in canalizead ana in polymorpnic, pyenotia nuclei. Tnese Late C-telophases give the aise appearance ot amitosis. Tnese recoastitutea nuc- cue le - Ciel are now tetraplola or,at-eny rete; are 2% with respect to tne paren tpl z Shag nuclei, Thus haploids can be maue to yiejd howozyzous aiploias. This prvcess TW? drsett arene of chromosome uoucling can ce repeatea indefinitely. In the reauction-aivisioz of the sporocytes ibevan ‘39) a fundomentally similar pattern appiies. In sone tic mitosis the presence of N-chromosumes ana the absence of tne spindle are clear diagnostic features eke colchicine eotivi ty au Te bese tr coed . [yl boeken, & . This mechanism ot potypioiday depends on the non-fune¢son or the spinaie and is not to te confused witn tnat induced in tne resting pnase by netero- auxin (Levan '39b)nor by that resulting trom tne adsence of new wasi-Toruavion uM. ruin pried wth ' . by purine alkaloias (Gosselin '40). Cytological investigations on tne e1rects OL colenicine, particulariy in- teresting to geneticists tor obvious reasons, nave deen wade On &@ great oumper ot organisms, pliant and animat, some of wnicn are Swiuarized in the foLiowing tabLe: TABLE I Investigations on cytoiogical etiects ot Colchicine. Material Efiect Reference tradescantia, stamen hrs c-mitosis Nebel & Rutivse 'S Biestiana & Datura poiypiotay Blakesiee ..'37 Bea, Aliium,eta root Genitosis Higsti '58 Alliwn root: @-mitcsis (gooa ilius.) O'Mara ‘ov Triticum root c-mitosis Reams & King '3e var. reots e-nitosis Shimamura '3y Pinus seeds mixed polyploidy Mirov..'39 Pteridophyte gan. polypLolay acsendani "41 Marchantia polypLoiay plakeslee '3yY Spirogyra binucleate Yamana,,'41 Saccharomycetes No Effect aichards '38 Schizomyeetes No Etfect Walker... '40 Review on plant material - Boas &VGistie ‘39. Tissue culture (an.) arrested metupnase Ludford '56 CLesrzia '97 7,.C. human cancer arrested metaphase Oughterson '357 vlouse organs aiter injection arresteu metaphase Brues & Conen ‘So Frog (tadpole) tail epidermis arrested metaphase Lein '41 Frog egg arrested cleavage Keppei .. '359 frog ese _ pyenotic nuclei Weish unpud Rabbit ova arrested cleavage,tetrapldy Pincus,, '3Y Orthopteran testis giant sperm, differentiation of celivuiar elements without Céli division Dogley "a1 Protos0m No Bxrect Beams «: Evans 'o5s,'40 TeratoLogical eisects,no acukt resuiting rrom tne aistorvion ot cieavage nave been reported as foliows: Oryzias (teleost) Waterman '40 various Galius (chier) Ancet & Latsemona *stropnosomic' Rena (anuran) JeLsh wapub. exogastruise ¥ ‘ . ! We have every reason to teileve tnat the ‘'arrestea metvapnase ot the Zoologists is cyuivaient to the c-mitosis ol tne botanists, The inninition otf Spindle function is the priire cause, , Se ak Wnise colchicine aemonstrates cemitosis witn tue ciearest aud great | est eriicacy, 1t is sy no means tne oOniy reagent wiat Wisi inauve porypiroiay 2? in tne manner aescribed. Tne #renen workers, particularity tne Gavauuens, Sone eee = “ST ee nev, anu Guinochet nave agescripeu , in a series Ot papers in tne C.R. Soe Bid a@ great mMeny Sucn gompounds, ineLuaing acenapinen, v-aichlorobenzene, najmnaien ana chlorc-naphthelene, prevyl-uretbane, ewuler,enioral nyarate, a@ua Obners, Mnere is no resolution of this 11St on tne spasis of cnewicai Surucvwure, indeed Luatora 'oo iinas tnat slignt moaditivations (auemetaysation) v1 wie coicnicine moLecuLe destroy its c-activity. In particuiar,however,tue anesStuetics are representea 1n tne 148i, and it nas been suggested that colchicine acts aS an anesinetic. *nis view is aGeait witn more fuliy bceLow. In concordance with the eiiects on spindte a great nuuver or con- sistent ocServations have been mace indicating wet py paysical correlative or we spindite inhibition is a modification or the viscosity fruy vow of mitosis Heilprunn '28). The ervect is atweys a reauetion in that viscosity, Wilbur * n tan Ua ay bee pn ftdbngahing Brg 6 "40 on Arbacia egys Tinds that the gsiati ir accompanying Metaphase is innib- ~— A Lived, tut that the stanaard viscosity is unatrected., Beams & King come £0 a similar coneLusion on Triticum seedlings, as does Wada ('40) on Living Treaes cantia Staminal hairs, anu Lein '4& on Ulycera eges in nypotonic seawacer, T N The causai retations or these phenomena are not revealed,nowever. That is to Say, is tne colunicine eifect directly on tne cytoplasmic viscosity, or is the Qiminution of the Latter an incidental consequerice to @ more ulrect innibition of the spindle. The supposed common benavior of anestnetiecs in aecreasing vis- cosity is interesting in this couuecsion, further prorress in inis alrection ls protatbly waitin, Tor inadependent progress of researcn on the nature of the spinale. Liliie('1l4,'18) has observed changes in waver-permeadility anda eiectricad —_— " polarity vf tne cell membrane auring sea-urenin egg alvision, Uowever,ao re- ports appear in tne 1iterawre witn respect to colcnicine e.ii6cts, Guinochet finds changes in tne pH and osmotic pressure oO: corcnicinizea Triticum seeurings.(G.,"46) 3 pH vacuole irom 4.8-9.0 to 4.4-4.8 oS decrease po ecytopiasm from 05.4 tO Dek 22 aecrease Osm Pr ‘cei! 7.1 to 11.1 Atmos. pby increase. The increase in osmotic pressure is a refiection of the very Swoiten ap- pearance that meristemutiv celis nave aiver etiective treatment with coleni- cine. The pi change is considered further below. There is as yet no rationale tor coichiaane e:.ects nere. The probien may however be attacked from a cnemicaL viewpoint. If only in a negative way. vhe WOrK tnere nas been more iruitiul. Generat—considerations on the chertistry of celi-aivision are aiscusseu in ute repers- The Biechemiesry of Celi-Division’ by the present autner,to wnicn _reterence gnouia oe made. Lt tne relationships boewvreen activity ena metaborisn ~-pere there ingicatea to pe-comptes ana obscure, the co1enicine situation can > only emphasizc this. Gal ('48) working on the anesthetic nypothesis tested the siiects of voi~ enicine on the activities or dehydrogenases, and cheered the resuLts agains? Wives «wi SOGium cacoaylate, wnicn has simitar cyteologicar eiiects, The Gacoadyiate nad no errect on any of the creis - sucrstrates. Colchicine had no erfect on succinic, glucose or giycogen dehydrogenases, but partialiy innibi- tea tactic ana cltric aenvdarocencses. The source,untortunateiy, was beet musele and Liver, in neither of which c-activity can be demonstrated. However an interesting nypotnesis to test woulda be tnat tne Lowering ort pH in Guinao- chet's Triticum 1S a consequence ot the accumulation ot Lactic ana citrie acid. This hypothesis is especialiy interesting in view orf Ruhlana ana Rams- norn's ('38) determinations of the R& in meristems, ana or tne presence or acetic dehyarogenases, indicating anaerocic termentation. This is aiscussea more completely in the succeeding report. At any rate, colchicine does not work cy blocking oxygen Supply at any ear- Ly stage. The etitects of oxygen bLock are very much airrerent. Brock, Druck- rey, and Herkin ('39) find that colchicine concentrations 00x those required for complete division tlock in Stongyiocentrotus have no erfect on the oxygen consumption of tnese eces. Patton & Nebel ('3@) tina that the drug does not atfect respiration at atl at .0002M, and nalves it at .cCOCO4i. Both of these concentrations probably are consiuerabiv in excess of that required for c-mis# tosis in tneir material, excised Zea roots, but no figures are given. The ans wer to this mechanism, Like tnat for the anesthegic inhibition ot otner cet activities is not to be sought in the total respiration or metabolism cut to some specific moiety tuereor. In spite of these negative conclusions,an enzy- matie hypothesis for c-activity remains a promising one, if not tne only one that is susceptible to any form of attack. Other observations on the enzymatic activity os coichicine are mostly negatives. However, Patton & Nebel, op cit, rind o aepression of proteolysis at a concentration of .COOQIM which may be at or not too much above tne threshola tor c-activity in tneir 4ea roots. Krugelis finds no inhibition of the acia-pnospnatase uctivity of young rat testis by colchicine. (unpub.) 7 Smith,'4l, ¢inas an acceleration of ma.t diastase, and no airect inhibi- tion of invertase by colchicine. Once again we can only say tnat no final conelusion is possible, 4. Although the main boay of this paper contains all ot the previous principal work on colchicine, tne author is in tne miast of sone investiga- tions which may yiela another approach. That investigation is ty no means complete: indeed it nas barely started, anda tnis Paper is not to ce regarda in any sense ave a puolication. It is intended ratner as a Sys tematization of the present situation, ana as a contricution tor the interest ot tTnOseE woo have shown some soLicituae for my exxorts in tnis uirection, consequent iy the remainder of tois paper will te on a more Speculative, nypotneticar basis. In the course ot some routine cytological stucies on c-mitosis in the Allium cepa root-tip, the author in Mareh 1y41 came across some inaica- tions of a gradient-of-suncepuicistiy vo cCorcuicine in the onion root tip. This was followea up by some work in the Spring or 194¢ whicn conitirmea this coneeption as rol.lows: Onion bulbs were grown in the dark over tap-water tor -4 aays untiL roots 1-2 cm iong appeared. Then a stated co1tchicine solution wes sutstitu- veu ror tne water, replaced every tew Aours. The root-tips were then cut ow av stated times, Lixed, and stained tor cytological stuay. Cornts were mae or 8u Longitudinai sections ot the types of division Tigures present, ana Ssegregateacinto tne groups: propnases,normus Me Tapnases, aueapnases, telcpnase ana c-mitosSes. tamera Luciaa sketcnes were maue under Low power,aivision tigures pickea out, and then ali ot these were examined unaer nigh pover,aw the type ana reiative locality of eacn sketch marked. Charts I anolII are grapnic representations ot the numpers of eacn type ot tigure in 2 sections with respect to time, aw 006% and .000% aqueous coicnicine respectivesy. ' The sketcnes indicated very aerinitely tat at tnese concentrations wne et tect Wag Localized, and that furtnermore, uormalr Waweks Were always round Wnen present,apicaliy to c-mitoses. furtuermore the Line of derarecation Was Snitted basipttaLiy at the higner concentration, altnouen vais Line shiftea very much more with time than in response to concentration aif- ference. to very satisfactory enalysis or tne cha:ts is yet tortneoming, ana wnecaata are stili tou incomplete to be entirery sipniztieant so tnaey are presented witout comment. There are atv Lex#st two possibilities in tue iaterpretation ot tne gra- Gient, requiring experimental test. Because ot dituerential apssorttion in tne regicn of aditierentlation or elonsationg chere ray exist an artitivias concentYation gradient aown tne root tip. vr tne cradcient may ve intrinsic 2 tne celis. In the rirst interpretation survives test by CUuUtIing anu GUuLe vuring experiments,it wili not be possible to use this approach. If the graaicnt is intrinsic, tne resuits of atvempts to extinguish or acventuate i “would ce most interesting. Such preliminary experiments with Pb, eyvanide, & methylene blue indicate only that the balance ct figure-types is upset by the accumulation of prophases vr of vegetative cells, in accordance with tt independent properties of tnese reagents. At tnis point one can Oly Say Ws trat experinents are being Planned and wiii shorily proceed, The authors attempts to measure dehyurogenacte activity on whole wvips Was vitiatved by the avsorption of methyaene blue by the cap and eLongavion regions,iirst,anad tren whe meristem, when tre Eucire tip was 6oLvrea,it wae cué Firs) tO aecoLovize in wie Tauncerg vaguw, anu teins GeGoLurigavion see Sted ULELsEcved by ule Presenree o: Corenigine., Tne eonaitions vo: we expere mens uengna repitigion, tae avvenpt then vo correiete coLsapse CL spira.e (inetien wish avini- Citic: Of SO.ig SOET OF ristanvile activity wen eonsinues, os ak yet is guste uneuccesstuL. One Lass importanc vonSideravion cugnt to ce Kepi tu mina: tue importance of distinguisning the asvrinsic 62; e0lS Ul curcnacine and tnose wnien oucur tarougn tie meuiatlon OF tne veseasvion oO: GeiL ulvision tne substance of tunis report was presented orally av a Bovany tee Seminar, Coiwipia University, on Aprii &¢,1942%, and ln written ioirm in a report to whe inssrucvor tor tuat course, Dr. 4G, Whaley. “he companion-report to tois : '™he Biccnenistry ot Celi-Division'’ was preparea only as @ vacKkgerounad 10r tue study oF cuichicine elects, ane is inoouplete to the exvent tnat tre cOLunicine propiem aces nou gCit on iv. July £0,1942, Josnua Leuerrerg O80 Hast 160 Street New Yorze,N.Y. O. iG BIBLIOGRAPHY Certain papers were not obtainable at CoLuncia University or tne Bot. G. Library except as abstracts, Ancel & Laliemonda "ay Beams & King "38 . ' 40 "40 "40 Avery,Blaxeslee.. "Si Blakeslee "Sy Boas & Gistle ag inege are Licted in tre avstract journe:, G.R. Soc Bio L503 Bioi Buil Biot But J.C.C.P, d.C.C.P, Awd+5es. J. Her, A.ww.B. Protoplasma o2 Brock,Druckrey & Herken '39 Aren exp Path u Pharin Brues & Cohen "36 Bioeh J 30 Ciark & Barnes "40 PSEBM 44 CGLearkin "37 J Path & Bact 44 Dooley "4d Am Mlicr Sow Trans 007105 Dermen 740 Bot Rev 6 Higsti "38 PAS Funner & Breipohl "35 Areh expmPata u Pharm Fyfe Ty CambriugetImp Bur Fi Br & Gen Gat "48 B.S. Ch.Biol. 20: Gavaudan & Gavaudan "Sg='ai Series in tne CRSB Gosseiin "40 C.R. Ac,Se. 2103544 Guinochet "40 CRAS 240; Heilbrunn "28 Tne Coiioid Chemistry or Provopiasi . Henry (ay The Plant Alkaioias 074-979 Churcniai-Lon Jacobson "25 CRSB 93 , Keppel & Dawsan 39 B,.B. . hein "AL CCNY Honors Report II, IIl 42 CCNY Bioi Rev May Levan "33 Hereaivas 24 "3Y Hereaitas <¢oasy '39b Hereditas 20:87 Litlie 14 J.B.oC. 1? 18 a Fnysiol 45 Lorthiori 55 3 oe Ludford ’ "36 Aron exp Zeliforseh 143411 Mirov & Stocxweil "39 oH. Nebel & Ruttile ‘98 J.H, Ougnierson,et al 137 PSEBM 6 Pineus & Waddington "39 Ja. Patton & Nebel "SY 28 Richards "38 J, Bact Rosendans "Ad Planta Ruhianad & Ramshorn "38 Pianta x8 - a Rushy,et al * 30 Props. ana Uses of Drugs o40-4¢1 Biakiston Shinemura "oY CytolLogia ; Smith "al Okt Ac Se Proce 212105 iad "4h Cytologia Wolter & Youranns 40 eSai . Wilbur 4 4 ve "41 Bio Abst ‘'4l :1ozl4e Yamaha & Ueda 1G0- i . soe” i Chart Chant me °o een nee Tc e a a Boho. 8 x1 w* | . | ra | x), ; eK x | | { { | | | \ \ Fag l A NWiam eepa 906% colchicine 3 hous Fig. Atlinm cepa ,005°'s colchicine ish ’ Figs. (and. bu Outline drawings of represeitalive scefions of tons Ouvien root- tips trated with thats held cane entra of eolchtetne. Tha tert apey io toward Hee top a, Hx pa The boy lamlines ae Hee peor ania aud the plane af demarcation biulcien, wicleas and e~ucliers nop cof: The sine arres art deuactd of He pinch ot afipeceecd Cerrvtag wonce Joell Any ane. @ indicates normed ag peng prophase q nd ieates hownsoh apparing putaplaar, arcap hen, KX indieates cmuitosis Dvawn at table level gf SoxX with the atid of a Core Jucida, J bedi hn 17 Y x7 Se { captions 14 Type.