RATE CHANGES OF THE MITO'I'IC CYCLE Thais Io: tho Dwru of Ph. D. MICHIGAN STATE UNIVERSITY Jack Van’f Hof 1961’ ‘ This is to certify that the thesis entitled RATE CHANGES OF THE MI TOTI C CYCLE presented by Jack Van't Hof has been accepted towards fulfillment of the requirements for Ph. D'degree in Botany -' fi/ééw Major professor A! ’/ 2 — 6/ 0-169 LIBRARY Michigan Sun University an Tllh C [l1 ilz'zrlyh Chz'xi‘lbilb 0f lilll‘: flIlUTIC CI;LL by Jack Van't Hoi‘ The purpose or this investigation Was to cevelOp anu utilize a relatively simple technique lor stuuyinb the effects of physiolobically active chemicals on the mitotic cycle 01 proliferating cells in a complex tlSSue. The experimental tools useo in achievinb the boal prOpueed were very youné pea seedlinps, colchicine, and restiratory poisons. The general methUU0105y involved treating the primary root meristem of pea seedlinbs for a period of 30 minutes with an appropriate concentration of col- chicine. The cytoloEical effect of colchicine is that of clumping or scattering of metaphase chromosomes ano the prevention of cytokinesis b; uisrtption of Spindle OrganiZation anu orientation. The final result of a short time treatment with colchicine is the proauction of a small, sbnchronously uividiné, population of tetraploic cells. This pepulation was subjected to 3 lb minute treatment with 2, 4-ainitr0phenol, 2, 4-dichlorochenol, Potassium cfianide, aLc potassium fluoride. by treatinb the population when it was in early interphase 01 the mitotic cycle, it was noted that both 2, 4—dinitr0phenol _ v- “a..d and 2, 4-dicllorophenol improved the synchrony ano eel ye ' - ~ -' ' whereas the appearance in division 01 the tetraploid cells, a breat deal or the synchrony was destroyed when cells were treated by these two chemicals at late interphase. Further, when the pepalation was treated with cyaride at either early or late interphase, no breat diirerence in reSpohse was observed. When the tetraploid cells were eXposed to z, 4-di- nitrophenol, a, 4—dictlor0ptehol or cyanide at the iiith hour after colchicine treatment, the second division of the population occarred precocioesly, thds indicatinb that treatment at this hoar Caused a decrease in the time re- quired to attain mitotic connetence. The natdre of the sySLem or systems responsible Ior the accelerated mitotic cycle appeared to be dependent on an increased rate of anaerobic glycolysis, since a combination of a cytolobically ineffective concentration of fluoride with either z, 4‘01’ nitrOphenol or cyanide prevented to a greater or 18886? depree the precocious second division or the marked pOpulation. 1mm; Clinifielt OP Till LITUTIC cyst; by JaCK Van't Eof A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree or LOCTCH CF rhltosGkHY Department of botany and.rlant PatholoEy 1961 aCi-ii. o“.. LLDG MM. Tb To his colleabues in the cytoloty UPOdp at :ichiban State bniversity, the author expresses his sincere bratitude for their aid during this investigation. To Dr. e.h. Wilson, for teachinb the principles of bio- loEical research and for his buidance and inspiration that were always present throubhtout the course of this work, a special expression of thanks is extended. The assistance of Mrs. hattelie hnobloch, hiss ASEPiQ Colon, hr. Roser Deuben, and Mr. Lcward oreenberb was ap- preciated for their part in preparinb and scorinb many slides. heknowledbment is made to the Rational Institutes of Health for defraying research expenses and for the financial support of the author and his family during his graduate studies. Thanks is also offered to the All-University Research Fund for partial support of research expenses. Finally, the author expresses his indebtedness to his wife, Nancy, for tactful encourabement and for aid in typing the manuscript. ii Ii. not; (3 TI Uh . bi ILiin TU m; in. v ILW . Q O O Q . . O O C O I § O O . MATERIALS 1113 b IUIJCLLULJS . o o o o e ObSEh‘VaTI CNS I DISCsSSION I OhEEaVATIUNS I DISCUSSION II CUhCLhiIUhS . SUMMARY . . . LI tiLI 0b Kn} HY . I Tie Technique. The Technique. . - Lffects or Hespiratory Poisons. hirects of hespiratory 1 He ! ‘ ' ' I' ‘ rf‘T lieiJLJtd b); {UK-1.1.1.14 Ho -» '1' o O O O s o a o o o 6 o o O 0 O O O O O Poisons O 0 O 0 szUL 17 Z2 c, .31. do '20 Thbm fault; I. The relationsLip hetmeen Colchicine Concentration, the number of aperrunt metaphuses, anu the number 0.1. Leti‘hplUiu Cells. 0 O O O O O O O O O O O O O I O 26 L 31 c: TLAI iloonhs iIDUhE fhvb l. f 9f; e v (n H, O 13. TheoretiCal curves oi the ratio oi oiirerentiat- ind cells to oivicinc cells versus time Ior oif- fegent systems. . . . . . . O O O O O O O O O O O O 6 ciabrwns representinb the cell cycle of microor- ganisms, the mitotic cycle of somatic cells she the meiotic cycle oi berm cells . . . o o o o o o e (.3 Model of the mitotic cycle. . . . . . . . . . . . . 1e Change in percent of effect prooucec C3 a 80 min- ute treatment with Various concentrations or col- C}liCinC o o e o o c o o o o o o o o o o 0 O o o o 0 Cu Tetroploic populations procuceo by various concen- trations of colclicine. . . "‘ o o o o o o o o o e o . LL/ Vazdnitio11
    " '" a. ““3 1 Plant Root meristem g" -—- Time Figure 1. Theoretical curves of the ratio of dif- ferentiating cells to leiOiné cells versus time for different systems. The meiotic cycle of berm cells is often used as a source of information concerning cell reprOduction. The events that occur in the meiotic cycle however, are not all the same as those of the mitotic cycle. a few examples of differences that exist between these two cycles ame listed below. 1) During prephase the chromosomes of dividing berm cells go through the complex manipulations involved in pairing and synapsis. The germ cell usually proceeds throubh two divisions P" V which result in the formation of four haploid cells. No deoxyribonucleic acid (DNA) synthesis taxes 01 place between divisions I and II of meiosis (Swift, 1950). 4) All maturing primordial germ cells have a dis- continuous cycle that is resumed benerally by either fertilization or parthenobenesis. A schematic representation of the mitotic cycle, cell cycle, and the meiotic cycle are shown in Figure 2. Between the beginning of one mitosis and the onset of the next, a number of syntheses must be carried out in order to prepare the cell for its division. It is of interest to the cyt010gist to know how long it takes a given cell type to attain mitotic competence and to know the sequence wherein these requirements are fulfilled. Aside from those experiments performed with single cells, most of the information concerning the questions mentioned hifferentiati Mitosis [:::] A“ /;I {j Mitosis Synthesis Synthesis Cell Cycle of Microorganisms Nitotic Cycle of Somatic Cells . ._u,i ..... i\ Division I of heiosis Synthesis / / NitOtiC VBiOtiC \ DiViSiOD II } of Meiosis cycle I Cycle ; t) ,' o / / a!!! ° Zygoto \ bertilization or __,,___,_‘,~.--_,_,_, .4 ‘ Parthenoéenesis Meiotic Cycle of uerm Cells Figure 2. Diagrams representing the cell cycle of micro— Organisms, the mitotic cycle of somatic cells, and he meiotic cycle of berm cells. above has come from the study of synchronous diviuino cells. Therefore, before consideration is biven to these questions, synchronous cell division will oe UlSCuSSBG. Some of the uQVuUtubbS of worxinb with a synchronized population of divioihb cells are that: l) the duration of both the total mitotic cycle and of mitosis Can be determined, 2) when performinb an experiment, all the cells I are more or less in the Same segment of the mitotic cycle at any biven time, C33 a synchronized pootlation represents a mass of cells that are relatively homobeneous hi their physiolobiCal state, 4) the experimehter's patience is not taxed as it is sometimes with unsynchronized cells (hubhes, 1952). The experimental cytolobist uses two types of syn- chronized cell division. The first of these is natural synchrony. Natural synchronized cell division occurs in the primary Spermatocytes of animals and in the microspore mother cells of plants. Caspersson (1939) used brasshOpper spermatocytes in his study of the role of deoxyribonucleic acids in cell division. hicrospore mother cells were used by Sparrow and Sparrow (1949) in their experimentation. Natural synchrony also occurs in segmentinb ebbs of hchino- derms and amphibians durinb the first two or three cleavabes. The advantages of using cleaviné eaus are expressed by heil- 10 brunn (1956) in his boxik, Th- D'iivninlt‘t or hlvll‘m ruo'l‘oru.sr«1. Another form of natural synchrony exists in tissue that is characterized by ra id browth and a relatively high rate of cell divisions Illfihufll a tissue a certain portion of the dividinb cells must be considered as beiné reasonably well synchronized thPUlbh several divisions. These cells are also homobeneous with respect to their physiological state, as well as to their position in the mitotic cycle. Taking I adVantabc of this homogeneity and making use of both a radio- active tracer anu autoradiobraphy, hUWard and relc (1951) l were able to label with Pad only those cells that were syn- a?” thesizinb DNA at the time of treatment and thereby marKed a population of synchronised cells that could be followed through the subsequent division. The work of howard and Pelc was one of the first in a series of experiments using autoradiOgraphy to study both cellular proliferation and cellular biochemistry. The use of colchioine in studying cell reprocuction is founded on the Same arbuments that apply to the use of radio- active tracers. Colchicine, because of its high specificity for cells that pass through metaphase (LeVan 1959; hadder and Wilson, 1958), will affect only those cells that are $0- ing through mitosis simultaneously at the time of treatment. Colchicine, therefore, affects only a relatively homogeneous broup of cells that are uivioir;b synchronously. The second type of synchrony is prouuced when unicellu— lar organisms are exoosed to a series of short time physio— loblCal shocas (neutzen and beherbaum, l9bs). Synchrony pro- duced in this manner is called incaced or unnatural syn- chrony. Induced synchrony has been produced successfully in amoeeae by James (1959, l 60) and in bacteria by Hunter- Szybalska gt a; (1986). In fact, this methoo has proven to be so successful that a number of systems have been develop- ed since its origin. Campbell (1957) mentions eibhteen sys— tems that are in existence and many of these are discussed in a review by Scherbaum (1960). In spite of its wide acceptance, induced synchroniza- tion does have its opponents. There is some uiSabreement as to whether or not the induced synchronized pOpulations of unicellular orbanisms are equiValent to cells in the natural state (Mitchenson, 1957) since synchronised cells are larber (Scherbaum, 1960) and have twice the amount of DhA per cell than that contained in cells oi a natural (unsynchroniaed) pepulation (Scherbaum, 1957). The question, tnoubh well founded, is rather impractiCal, for the udVantabeS of syn- chronization greatly outweigh the disadvantages. The duration of the mitotic cycle in any particular case depends on a variety of factors: for example, Laughlin (1919) found the duration of anaphase in onion to be temper- ature dependent and brown (1951) observed that the mitotic Cycle time of pea root cells varied with temperature. Fur- ther, Frankland and Ward (1895) while meaSuring the time be- tween fissions of a schizomycete, Bacillus ramosus, found that cell division and growth were influenced by light and 12 temperature. The duration of tie mitotic cycle 01 cleavinO raboit 6&58 was measured by Lewis and orthory (1329) usinb photo- Lraphy. lfilunus (1930) also followed the cleavave of rabbit ebua. thumWay (1941) has reported the cleavabe time in fro5 e5bsand nubh (1948) cites the ClUuVQ$C time or other am- phibians. aeutren (latl) rrcoruee cleaVabe time of sea ur- chin 6555 in measurinb IluCtaatiOnS in oxygen consumption durinb the mitotic cycle. Unlike cells of hibher orbanisms, the cycle time of microordanisms usually is a matter or minutes rather than hours. This time Can be determined by induced synchrony (Zeuthen and Scherbaum, 1954) or by measuring cell number in- crease durint the log-phase browth period (Mealoe and Lark, 1954). In studyinb the respiratory chances duritb mitosis, Stern and Kirk (194a) have been able to use anther size to determine the LlVBI’uLL stauc or contained microspore mother cells. Time in this case is a rather poor indicator. hever~ the less, time in most instances is the more practical par- ame ter. ‘arlson and Hollander (1946) timed the mitotic cycle of the grasshOpper neuroblast and found it to be three hours twenty-cibht minutes. Interphase lusted only tuenty-seven minutes and proved to be most sensitive to ultraviolet radi- ation. Fell and hubhes (134a) combined phase contrast mi- croéraphy and cine-photomicrography to study the mitotic cy- cle in mouse cells. They observed eibhteen intermitotic times (interphase) and loune them to rance from b to lb hours with tre moue bein; at a hours. microspectrOphotomee try, in addition to photometry, was useo by walker and Yates (1952) in their study of DNA synthesis. They found that the interphase of chick heart cells brown in tissue culture last- ed for 10.7 hours and that the entire mitotic cycle time was 15 to 16 hours. tray and Scholes (1951) calculated the mitotic cycle time 01 Vicia faba by delayinb cells located in the x-ray sensitive seomtnt of the mitotic cycle with x- radiaticun Usinb til.ioh1y maiingAtical tunxroach, they found the mitotic cycle to last from 15.4 to 25.4 hours, with mi- tosis being 2.6 hours and interphase beinb 16.5 to 22.8 hours. These values were corroborated by Ecuard and Belc (1951) using the autoradiocraphic technique and also by Taylor, hoods, and hubhes (195s) who found octaploid cells after 56 hours of continuous colchicire treatment. In order to be octaploid, these cells hae to complete two mitotic cycles, thus givinb an averabe mitotic cycle time of 18 hours. Colchicine Was also used by Evans 52.3; (1957) to de- termine the mitotic cycle time of Vicia faba. In this case it Was reported to be about 24.5 hours with interphase beinb 20.7 of these 24.5 hours. 'lritiated thvmidine has been used by timber (1360) who studied the mitotic cycle time of Tradescantia root tip cells, and by guastler and Sherman (1959) in investibatinb the Vari— ous segments of interphuse in the mitotic cycle of mouse. Rudiouctive isot05es Leve beon used by Lornsey uml honerd (1956) she Painter'unu hrem (less) in similur studies. A review of the production uzzo use of tritiuteci cozr.pour1o8 by Taylor (isco) but recently been puclishee. The questions, “Let ere the requirezzxtnts 101‘ mitotic competence gnu in shut oruer these requirements ure fullilleo, muy be considered simlltuneousiy. The use of P52 uno tri- tiuteo thymieine plus the uutoruuiogruohic technique Les ulloweo the fructionuticn or interchuse into bl (u kerioq immediutely followinb telophese wherein Dhn synthesis noes not tuke clique), the S stuée of interpnuse (wherein DNA syn- thesis noes tuKU niece), unu ”2 (a pCPlOQ ioilowinb the 5 stage and Just preceeoiné mitosis wherein no Dhn synthesis takes place (houuro und Pelc, 1951; tonnes 32 e1, lJOO; guestler uno ShCfiWkfll, 1959). The term ”antephuse" Wes pro- posed by Bulloubh (1952) to apply to the state of relatively low metabolism that exists in cells Just beiore mitosis. It is yrobsblc thut 52 and "enteehuse" use synonyms. The synthesis of chromosomal protein nus resorted to occur curinb interthase simultaneously with Dhs synthesis by QC _ _ ‘ 1 u u 1 . . hoWsro and Pele (1352). They uses 5 anu the uutorudio- brush technique on the cells oi the root meristem of Vicie '/ L fsba and found the 5" to be accumulated ubout the sums 52 time as P which wus usec to measure DNA synthesis. More recently Frescott (1903) measured DNA synthesis, (nLA) ribonucleic ucie synthesis, unu protein synthesis in syncironizeo Vopuletio s o; letrehhmcnu une round DEA syn- thesis to he nonlinear uhU to oceur first followinb division. ANA synthesis also occurred et a non-linear rate out did not reuch SibniflCaECC until Dhn synthesis mus comoleted. Most of the RNA Wes synthesizeu JJSE before oivision. Protein synthesis on the other heno proceeoeu at a slow linear rate ourihb the entire interyhuse. rrotein synthesis therefore, occurred simultaneously with DNA enc mks sbnthesis but never at the some rate. The subjects of colluler enerpetics, cerbohydrute me- tabolism, ind respiration hate been investibeteu for many years. Tie work done by Ewenn (19t4) indiCQLed a CO libht reversible inhibition oi cleuvube o1 merine e505. twenn cis- covered that there Web a time beiore which CU would inhibit the cleavebe immediately I'ollowinb tPCathnt. If treatment were performeo after this time, only the second cleavabe followinb treatment coulu he inhibited. SWunn interyreteo these reSults us indicating the existence of an “TB (ede- nosine triphosohute) Stureue mechanism which hso to oe at a Certain lele before cleaVuLe could proceed. he further hypothesiacd that this ATP storaée mechenism woulo have to be PEplenishec iollowinb each cleavage. This concept is SUp- ported by the work of Clowes uno Krahl(1957) and Krehl end Clowes (1937) who observed that a rich respiratory rate due to uncouplinb resulted in the inhibition of clesVabe in the sea urchin egg. They obtained similar results with both ni- trateo and hulocenated phenols when these compounds were used id to increase oxyten uptake. hamburber ane heathen (19:9) also found that DNP delayed the division of a synchronised poyu- letion oi Tetrahygena. Similar reSalts were observed by Immers and nannstrom (1960) when they treated sea urchin cabs for only 30 minutes with a very low concentration or DNP. The imoortance of carbohydrate metabolism is empha- sized by the work of pullough (1952) and wilson, Iworrison and Knobloch (1359). These workers retort that the addition of a Caroon source to a competent system of dividinb cells lOCateo in antephase will increase mitosis, thus suggesting that antephase is somewhat dependent on a carbon sodrce for the entrance into mitosis. a compilation of the obserVations resorted concerniné the mitotic cycle allows the construction of a maiel which repr‘sents most oi’vdnit is Known to occur Marina a sinble re- volution of the cycle. Such a mooel is illustrated in Fig- ure e. It should be pointed out that this model is not meant to represent absolutely the relative time of occurence of a Biven synthesis or reaction. f*> Interphase ——{~—Mitosis~——’ 00 7| 00' 51 DNA Synthesis 6‘ a; RNA SE. frotein :"nthesis high Carbohydrate Metabolism Figure 5. Medel of the hitotic Cycle Imi'l‘anlnlb hi'xl," sinuous ulll‘il’slmb mliuIlmI-fl‘nl‘. PliO’o'lal/Unls The tissue used in these investigations Was the brow- iné root tip of risugvsativum Var. alaska. ihe peas were furnished by the berry-morse Seed Company who took pre- 1 cautions to make sure tne seeds were disease-free, of relative genetic homOgeneity, and had not been treated with any anti-fungal agents. The peas used for experimentation were soaked in cis- till d . ° 1 0 0‘ '"* - " e water Ior Six hours at e5 0. lhey were then rolled in paper towelinb and moistened. hext the peas, rollec in paper towels,were placed in an upribht position in beakers containing about one inch of distilled water. n sheet of waxed paper was then wrapped about each rolled towel after which the peas were allowed to berminate in a berminator at 8500. Germination continued until the pea seedlings had roots that were between two and one-half to three and one- half centimeters long. at this time the seedlinbs were col- lected ior treatment. seedlings with roots of the cesireo lenbth were sue- Peneed on waxed, one~quarter inch wire mesh above 600 ml. beakers that contained one-fourth strenbth hoatlanc's .. - ,- ,v; x” 4‘} nutrient solution. The contents of one-quarte. strenben Hoaglanc's solution are listed below in Frame per liter. . ,. -H.“ ‘l i... CEA ed in ration between paper towels. The slide was then plac " ‘ l “’0 an alcohol solution of 90% tertiary butyl alcohol and v/ ethyl alcohol to dehycrate. Followinb dehydration the slide was made permanent with dianiane. F17 '+"’ a v ./v ‘ -‘- " ' ileatment solutions were made iron the Iollowinb chemi- cals: H 3 2 C / C \CNOZ c/ \oc, C C C C Noa c': 2,4-dinitrophenol, 2,4-dichlor0phenol, potassium fluoride (HE), and potassium cyanide (new). The source of DB? was Eastman Chemical Company; DCP was pur— chased from Light's Organic Chemical Company; KB and KCN were obtained from the Baker Chemical Company. The DNP solutiOns were prepared by pourina heated cis- tilled water into a flask containing a small amount of DNP. From this solution the desired concentrations were made by diluting with distilled water. In all the experiments util- iZihb DNP, the concentration was 4.3 k 10—5 M. Solutions of DCP were made by adding the weighed amOunt 0f UCP to a flask containing distilled water. This solution was then placed on a matnetic stirrer to assure complete dis- SOlution of the DCP. Treatment solutions of DCP were pre- pared in the Same manner as those of UNP, the one exception being that the treatment concentration for DC? was 4.6 A 10.4 M. Kl" and KCN solutions were mace in much the same manner as those of UMP and UCP. The use of KC‘i necessitateu the use of the hood for all its handlinb including the eXperi- mentation. When exyeriments w ;re heinb carried out in the hood, environmental conoitions were equivalent to those of eXperiments not cornice Ltu in the ”noon. '. "1 £2 ta;Si.A},J.I«)£&, I hithin the meristem or a growint pea root, there is a fraction of the cell pOpulation that is revolving in the mi- t0tic cycle. In the presence or an effective amount or col- chicine these cells as they pass throabt mitosis will have clumped and/or seattered metapiases (hadoer and‘uilson, IBbe). Some of the cells having auerrant metaphases will in the followinb mitosis be tetraploic and therefore distin- guishable from the cells not affected by the colchicine which remain diploid. In the pea root, diploid cells and tetraploid cells contain 14 and Bo chromosomes respectively. These facts present the possibility of pPUUuCinb a te- k? traploid population oi cells by treatlnb the meristem with colchicine for a short period of time. The tetraploid or marked cells should be only those that passed throubh meta- phase during the time of treatment. These cells should also be relatively homoheneous with respect to position in the mitotic cycle and consequently divide somewhat synchronously in subsequent mitoses. To test these hypotheses, three experiments were per- formed. The iirst is composed of two parts. fart one was carried out to detennine what relationship existed between the concentration of colchicine and the degree and duration of effect. Figure 4A shows Lraphically the rates oi appear- ance and diSappearance of clumped and scattered metaphases followinb a 50 minute treatment with colchicine concentration of 6.26 X 10'4 M (curve A), 5.63 X 10—4 M (curve 5): 5-0 A L) g) ( 1 r_- ogwoagoaoo a w:0 & p>.a A......vmm 023238 2 IS a an; Till; a 0:3338 2 Te a 05. hills 0 023238 2 “-3 < 3:... Till a oCHOH£oHoo E vuoa A om.o A.llll|; < .ocwoflfloaoo mo menaudpp .odwoazoaoo mo nCoodoo msoand> Qua; pdeEpsopp machowapnooCOo mjoflan> he ooosoomm opscfis on a an voodooam ooomwo ndoapdasdol oaoamsnooa .nv majoan mo psooaou CH onswsu .zv ohsnflm mono: mason 3 .3 3 ma 3 n s o e m. 0 tall 1 d i . liq u u 4 ./...d.IeIHI./-r.ooooo c\\l fit 40H / / .v / I./... o\ 4 N. I at l ON i wa low . .om - S 1 8 L lob ism - on g .2. POOH t I L,‘j 10'4 M (curve C), 4.38 X IO"i h (curve 0), and 3.76 X 10-!1 n (curve h). Since the rate of appearance of aberrant metaphases is very similar for all concentrations used, little attention will be biven to this part of the data. On the other hand, the recovery curves or the rates of disappearance of aberrant metaphases Show a great deal of difference. between one and two hours the curves begin to reflect the different amounts they represent. The number of abnormal divisions is already decreasing at this time for 4.53 h 10"4 h (curve b) and 5.76 k 10'4 M (curve B), while the three hipher molarities, thOUah not QGCP8&Sin6, show no net gain in clump and/or scattered metaphases. At three hours all five concentrations show either a decrease or no net increase in effect. From the general pattern of these recovery curves it can be said that the rate of recovery from a 30 minute treatment with colchicine is more or less inversely prOportional to the concentration. It should be mentioned that the curves as shown in Figure 4A and 45 have not been subjected to statistical treatment to demonstrate a significant difference between them, since the existence of a statistical difference is ir- relavent to the more practical purpose of the experiment. In addition to testing the two hypotheses mentioned pre- ‘yiously, the experiment was also performed to determine what concentration of colchicine would produce a tetraploid pOp- ulation of a usable size and a minimal distribution in the ««t._ NU mitotic cycle. A pOpulation having these characteristics should be easily distinguished because of its pronounced maximum and minima. Further, colchicine has a cytolobical effect that changes both qualitatively and quantitatively with dose. These chanbes have been taken into account by hadder and Wilson (1956) in their analysis of the colchicine effect on pea root cells and by hyppio (1954) in his oc- servations on the residual cytolobical effect of colchicine after short time treatments usinb the same material. The cytoloEical effect of colchicine, as indicated by these studies is statistically analyzable during continuous treat- ment, however the recovery curves observed after a short time treatment are not mathematically intelligable if both the qualitative and quantitative changes are taken into ac- count. Figure 4A represents only the quantitative change after treatment and therefore does not show entirely what is happening. On the other hand, a plot of the percent of clumps and scatters abainst tine appears to be the only lobical way to illustrate a correlation between the number of cells affected by colchicine and the tetraploio cells originating from the affected cells. The second part of the first experiment concerns the measurement of the number and distribution of tetraploid cells produced by the 50 minute treatment with the concentra- tions mentioned above. Figure 45 represents the results of these measurements. When considering the general pattern of the curves in Figure 4B the followinb characteristics are apparent. 1) All the curves have a maximum at twelve hours. 2) The curves are more alike from eibht to twelve hours than from twelve to eibhteen hours. 03 The distance between the first appearance of tetra- ploids at eight hours and the first minimum is ap- proximately prOportional to the molarity used. 4) The area under the curves between eight hours and the first minimum is also somewhat prOportional to the molarity used. Moreover, a comparison between the curves of Eibure an and the curves of liEure as show that the number of tetra- ploids is proportional to the number of aberrant metaphases and that the distribution of the tetraploid pOpulation re- flects the rate of effect and the rate of recovery from a treatment with a biven colchicine concentration. The slower the rate of recovery, the greater the distribution of tetra- ploids. To determine more accurately the nature of the re- lationship between the number of abnormal metaphases and the number of tetraploids, the data shown in Fibure 4A were ex- pressed as the number of affected cells per thousand cells. The conversion was done in this manner: the average mitotic index (the number of QiVidiUE cells per thousand cells) in the pea root was found to be about 70. One-half of the 70 dividing cells were post-prophases; therefore, 100; on the ordinate of ribure 4a is equivalent to 55 aberrant metaphases. 1,”? The use of the proportion, iggé as %& , where 1 is the percentage as IYHMijfURm figure 4n and A is the UuMLéf’Of af* fected cells per thouSand, completes the conversion. further, since the polyploid index is epr€Sb ed as the number of tetraploid cells per thousand cells, consideration must be given to the relative contribution of QlVidlng cells to the thousand cells after one complete revolution of the mitotic cycle. The affected cells do not undergo cytokinesis, hence they contribute but one cell to the total pepulation of cells in the meristem. The unaffected cells however, do undergo cytokinesis and therefore each adds two cells to those of the total meristem. The ratio of cells contributed by affected and unaffected cells is then % or 0.5. The net result of this difference is a dilution of the tetraploid population by factor of 0.5. Taking into account this di- lution factor, the followinb relationship exists between the affected cells and the tetraploid cells. Du N (Ht—51) (0.5) = (P) ( JD) Um Where P is the probability of a cell completing a revolution of the mitotic cycle, Na is the number of affected cells per thousand cells observed from time tO to time t1, NC is the number of tetraploid cells per thousand cells counted between eight hours and the first minimum as shown in Figure 4b, om is the length of time a biven cell is observed in mitosis, (3 u to is the time the experiment Was bebun, and t1 is the time the recovery curve has a value of ten affected cells per thousand cells. bm for the affected cells is approximately 1.5 hours, while bm for the tetraploid cells is three hours. The dif- ferent values for Dm are due to the fact that colchicine af- fects only post-prophase cells. Therefore, the amount of tine a given clumped and/or scattered metaphase can be oe- served is 1.5 hours. on the other hand, tetraploids are counted in all stabes of mitosis and thus are observed throughout the three hour mitotic period (Hyppio, 1954). Calculations for five molarities of colchicine are 5iven in Table I. Table I. The melationship between Colchicine Concentration and Na, {3, and “t CUUQLNTMATIOh ha fig t, d 6.26 x 10'4 M 600 500 295 5.65 x 10'4 r 554 267 250 5.0 x 10“4 Is". 467 255 165 4.58 x 13'4 M 255 141 125 5.76 x 10-4 M 250 140 116 The data listed in Table I subbest that a one to one relationship exists between the number of affected cells and the number of tetraploid cells. At higher concentrations this relationship is excellent, however at lower concentra- A) l y. 1 r tions there is less abreement between the data. This lack of agreement is probably due to the fact that a greater pro- portion of affected cells are soattered metaphases when lower colchicine doses are used (hadder and Wilson, 1955), and the probability of a scattered metaphase becoming a tetraploid cell is much less than one. (a scattered meta- phase is less likely to include all of the chromosomes in the reconstituted nucleus because they are distributed throuéhout the cell. If a reconstituted nucleus does not include all the chromosomes it probably will not divide again). On the other hand, the probability of a clumped metaphase becoming a tetraploid cell is almost one (hyppio, 1954). The second experiment performed to test the hypotheses was to treat pea seedlinés with the same concentration of colchicine for various lengths of time. The molarity used was 5.0 x 10"4 M and the treatment tines were 10, 15, 20, and 25 minutes. A secondary purpose of this experiment was to determine whether or not an improvement could be made on the rate of recovery as well as the production of a tetra- ploid population of appreciable size and limited distribution in time. Figures 5a and 5b show the results of this experi- ment. Figure 5a indicates that the percent of affected cells is increased with treatment time and that the rate of recovery is approximately inversely pPOpOPEiJHal to the duration of treatment. bigure 5B indicates the possible existence of a threshold O®~r HO 0 1.13) ... .p a. p:o-+¢ip opjzfla on. 7}; one tamoaoo 10H s on new; passions: opoufia .9». Allow 3: A225 .0H A. .8 no. A. w 5:5. .01. Alloy X: 7.25 m as. .onflowzoaoo 4 noa .4” 01m m3 voodoonz noaosajdom dadsldhpoa .nn enema; poowqo we newomfiamw .mn eaooam mason mason m .3. n o lidaHlal 1H wOH c. N W O 4-0 o o o o . ooo+0'oooo-. .p...\ / O \I. ‘1‘ . L N. a L om o T. 0A d .L 1 ow 0 TL. D. o a E m. Joe 1 n WW 1 0m and a om L 0e 1 ow . om .ROOH "/ ,9 peasiqaog JO padwnrg sessudomd-1904 51 that must be exceeded in order to proauce a population of any notable size. The threshold in this case was exceeded only by the 25 minute treatment. The third experiment was desibned to determine whether or not a tetraploid pepulation would divide somewhat syn- chronously for three revolutions of the mitotic cycle. Figure 6 shows the experimental results. The pOpdlation illustrated in figure 6 was formed by a 50 minute treatment with 5.76 X 10'4 m colchicine at zero hour. This population divided synchronously for at least three revolutions of the mitotic cycle. The synchrony, however decreased with each revolution as indicated by both the lowerinb of the maxima and the increased dispersion with time of the tetraploid cells. Indeed, if a curve is drawn from maximum to maximum and extrapolated to include at least three more mitotic cycle revolutions, it appears as though no synchrony remains after this time. At this point the marked population would be equally distributed throubhout the mitotic cycle. The appearance of two maxima durinb the third (III) division is another interesting observation. hvidently there is beginning at this time a separation between cells that are revolving about the cycle at different speeds. The first maximum being the faster cells; the second being the slower cells. Figure 6 yields another very important piece of in- formation. The distance between the maxima of divisions I, II, and III averaées twelve hours and since each division r. -« .l; 1 ‘JL/ audmmp cpdsae on m an ccQSUOAQ .eaaoaeaaoo a e-oH A e>.n sane acme soapsasmoa afloamnhpmp m mo msonH>HU omega .0 madman mhdom. . 0v mm em en ma ow mm m em NN 0H 0H VH NH OH x 1.11 - ltfi I q 1 4| in! 4| 1 h H u q - m 4m HHH HH H -ma 43H LQH new be V i L"; .‘CTOJ 510* O Xapul represents a complete rcvolution of the mitotic cycle the twelve hour averaoe is LLB total cycle time or the meri- stematic cells in the browinb pea root at 22.506, which is in agreement with brown's (lfltl) estimation of the cycle time in the same material. EL; hdY U} onSLHVhLIULS I l) hoses of colchicine CXCEBUlDD a threshold will pro- duce a tetraploid pOpulation. 2) The size and distribution of the marked population is determined primarily by the rate or recovery from a biven dose. L) The rate of recovery from a treatment of colchicine is approximately inversely proportional to both the concentration and the duration of treatment. 4) a marked pOpulation of cells will divide synchronous- ly for at least three revolutions of the mitotic cycle. 5) The mitotic cycle time of pea root meristem cells is approximately twelve hours at e2.tOC. JILCILSIUL I Oribinally two hypothScs were made CUNCCPDlLb the use of colchicihe in markinb a naturally synchronized pepulation of cells. These hypotheses were: (1) short time eXposure to an effective concentration of colchicine should tab a broup of cells that pass throubh mitosis durinb the time of treat- ment, the distinbuishinb mark beinb a doublinb of the number of chromosomes and (2) the marked pOpulation should divide somewhat synchronously during the successive mitoses. The experiments performed to test these hypotheses in- dicated that tley are reasonably correct. In fact, the data presented in Fibure 6 ahpPOuChGS the ideal curves of Cuastler (1960) for systems having a constant speed of proliferation. This is probably due to the fact that colchicine affects only a small portion of the mitotic cycle of a cell. In pea the duration of metaphase is between 24 and t4 minutes (brown, 1961; iyppio, 19b4) tierefore, the colchicine is eifective durinb 1/60 to 1/13 of the cycle time. This mibht be compar- ed with the use of the deoxyribonucleic acid (DNA) labeled with P32 3 or H —thymidine. In mouse intestine cells the bha synthetic period is about 7.5 hours and the total cycle time is £1Pproximately 19 hours («UaSBlbfy 1953)- The fPuCtiON 0i cells labeled would.then be 7.5/19. In Tradescantia root cells this fraction is 10.8/20 (timber, 1960) and in mouse Elrlich ascites tumor cells it is 12/18 (Hornsey and HOWaPu, 1956). It is suSpected that the small fraction of cells labeled by colchicine accounts for its approach to iceality. ’/ QJ' “ x CD The loss of synchrony and the continual randomisation of cells in the mitotic cycle Ls also illustrated in Libure 6. These two plehomcna are by necessity inversely propor- tional to each other. For eXample, if continual randomisa- tion did not take place with time the distribution oi the marked population would be similar to the distribution of the affected cells (Fibure 4“) for all successive mitoses. Obvi- ously this is not the ease as inoiCated by libdres 45 and 4b and Fibure 6. nandomiaation is primarily due to cells that are re- VOIVlDd in the mitotic cycle at different speeds. This dii ference in total cycle time may be due to an over all slow- ing down of all the processes of the cycle or it may be due to a delay in some particular segment of the cycle (ouast- ler, 1960). In any event, randomization does take place in the pea root and one of the factors aidinO randomisation is a dif- ference between cellsrevolvinb in the cycle at different Speeds. This is evident from the fact that the population shown in Figure 6 has a lower maximum with each successive mitoses and therefore a wider base between minima as well as a beneral separation of fast and slow revolvinb cells as shown by the two maxima in division III. In spite oi the effect of randomization, the technique developed by these investibations does present an excellent method for studying the different stages of interphase since synchronization lasts for at least three division cycles (ribure 6). Measurements illustratinb the cifrerenccs that occur curiné interphuse are sibnifiCant only in terms of their relationship to the controls. These differences however, are real and repeatable as will be seen in the followinb discussions. OLSimvnTIoNS II The physiolowicelly aClee chemICels used to investi— 5ate the hypothesis that differences may exist in cells which are in different sebments of the mitotic cycle were those that may be classified as reSpiratory poisons. The first of these Wus 2, 4-dinitr0phenol (but). DNP Wes used for a number of reasons, some of which are: l) The cytolobicul effects of oNP had been well described by Mdhllné gt El, (1960). for the pee root cell. These effects are visible end measurable with the alQ of u libht micrOSCOpe and are represented by an unusual accumulation of late-pPOphuses. 2) The cytolobiCul effects of LNP are reversible et non-toxic cnncentrations (Muhliné gt El, 1960). b) The physiologiCel “no biochemiCul effects of DNP are relatively well Known (Simon, labs). 4) DNP affects probably one of the most basic processes of living protoplasm, numelg, the process of bioenerbetics. Therefore, in se- lecting a chemical capable of showing the ex- istence of ph3510105iCd1 differences in the various cycle segments, our seemed to be the lobiCul choice. The first experimeht utiliZlnb DNP was carried out in r‘ y] D the followinb JWJHMeP: u tetraploid populdtion wus formed by treutinb pea seedlinbs for LO minutes with c.7e h l0"4 M colchicine. Four hours luter, by which time nearly all the marked population Was in early interphase, a sinble broup of seedlinbs Containin5 u marked pOpulution wus exposed to 4.5 k 10-5 M DNP for 15 minutes. A similar broup Was treat- ed five hours after marking, still another at six hours, and etc., up to nine hours after colchicine treatment. By nine hours the tetraploid pOpuletion huc passed throubh the interphuse seament of the cycle und was ulreedy enterinb mitosis. hipure 7 shows the time of appearance and the distribu- tion Of the untreated control population, the pOpuletion that Was treated at four hours (eurly interphuse), end the population that Wes treuteo at 8 hours (late interphese). These curves inUlCate that a difference does exist between early and late interphuse. The tetraploid cells that were treated at the 4th hour snow both a two hour delay in appearance in mitosis and a decrease in distribution of the murked cells. On the other hand, even thOubh the eibht hour curve also shows a two hour delay, the pOpuletion has an increased distribution. Ir addition to these observations, Fiéure 7 SUébBStS that in order to Carry out investibutions involvinb two cycles, only early interphase treatments would be pructiCsl, for too much synchrony is lost if UhP treatment is given when the population is in late inter- phase. .Jcflxmse bogus mp 05 o. .v .Honecoo A. .Jzflxhsa Lopes majog Adoa ooosohp Coaosajmom ebsa Lo wages :H douseho mcoHpsHJLQQ Jfloammpuog . (/9 HJHe ecesohp sowptazmom Axt..lxv A0.....ov .LAJ Jews endgahousfl Mo coamfl>fld pahwa one .5 eLShHm maze: om 3H 0H ea NH ifi 1 'Ci ‘ q O) H m xobul otoIdeoa “at: H 3 r, Because it was noted that a 15 minute treatment with DNP at 4 hours resulted in a two hour delay in the appear- ance of the marked population, the question arose as to whether or not a series of 15 minute treatments at two hour intervals would cause the tetraploids to be delayed lonber than two hours. To answer this question the IOLLOhiUb ex- periment was desibntd. boar brodps of seedlinbs were used. The first was the control, the second was treated at 4 hours after marking a pOpulation, the third was treated at 4 and 6 hours, and the fourth was treated at 4, 6, and 8 hours after colchicine treatment. The eXperimental results are illustrated in ribure e. These aéain stow a delayed appearance of the treated pepd- lation as compared to the control. (bibure 8 does not con- tain the results of the 4, 6, and 8 hour treated population because, as stated previously, a population treated in late interphase is too dispersed for any useful measurement to be made durinb the second division). again the distribution was decreased by treatment. There are, however, two addi- tional obserVations that are of major importance. These are (1) the delay in the first division is no greater when a pOpulation is exposed twice to oh? and (2) the pOpulation that was treated twice with DNP appears on the averaGe two hours earlier in the second division than the single treated population. Moreover, the cycle time for the 4 and e hour treated tetraploids is of the order of a hours whereas, the cycle time for the control and the 4 hour treated cells a : ‘iJ. .wsaxmde Longs mason 3 pcs e um ma; Lows Condos» Axn.lllé .ecflgass new «a. wage: as 9.3. goats wooden» A0. ....ov Joyocoo Atlulls .mCOHpsaijQ afloadsnoep Ho mzoamfl>fld esooom Use pupae .m endows madoh mm mm vm NN Om 3H 0H ea NH OH a q q 11 q q '4 4| ‘4 # J1 . L") (3‘2 xenuI pIOIdKTOJ 12:, is about 10 hours. Therefore, it may 33 stated that the duration of tdm::nitotic cycle between divisions I -nd II was decreased bv the double treatment with DRE. Naturally, a number of Questions arose concerninb the Cause of the effect exhibited by the double treatment. One of these involved the necessity of a double exposure in early interphase. The accelerated cycle was possibly the result of treating a group of cells lOCated in a particular part of interphase that Could respond differently than :1. other interphase cells. The argument seemed probable, LUr cells that were exposed twice may have resumed reVolvin5 in the cycle after the 4 hour treatment, and thus occupied a different sebment of interphase wnen the 6 hour treatment was biven. To answer these questions, an experiment was deSlbncd that involved the treatment of different pepulations at 4, h _ ) .» ‘1 _ “ ‘1‘“ ‘ . 'A .2 . .111 5' ““0 0 hours. The expeiimental lesults ale Shown l Fibure 9A, 96, and 9d. The results of a similar experiment are illustrated in Figures 10A, 105, and 100. In each case, the experiment was carried thPOubh two revolutions oi the mitotic cycle. It is evident from these data that only a Sinble treat~ ' -' " 3 ‘me and ment with DNP is necessary to decrease the cycle ti - . : ._: H 1103.]? further, it is apparent that the cells in the filth ' ' - ‘ ‘ a‘ ‘l- sebment of the cycle are the only cells that show an ece erated second cycle. ‘ "* ‘“ ‘ lls The effects of a short time treatment with our on ce mkdog Nu £N ¢N on ma NH m — u 1- I u n 1 4‘ I d a \I coasoaa ?-Icl|¢ Heepcoo ATIIIIIL .oqaoflsoaoo spa; mcflxane Lopas Auoa ossofiav maso; 3 3:3 .Aza ohjnflav mason n .Ain otsoflaV mason v pm me: E gnoa 4 a.v npfls psoEQmoho oodzflfi ma m Ho pooaqo oge um madma, ma o mpso: DN vw ON 0H NH 3 a [N \ - . 4 q mason om em on 9H ‘ 4 vepuI ptoIdKToJ v) 03 xeouI ototdfltoa :0 O3 xepuI ptoIdKTOJ oopoohe ?¢|Ilov Homoeoo Avllllov .ocaoflsoaoo Spa; dnoxass Logan Aooa ossowav mason a one .AAOH ohsoflav mpdoq 3 .Aioa enjoflav mhzoz v on «a: a 10H K @.v Low; u a ocospmoap oajzae 3H a ho pooaao one (1 Q 30H mesons mesa; us he. an om ea ma 3 14 I a c a I I I I J‘ o to (D xepul DIOIdKIOJ mhjo: mesa: OOH 0L” Swww m 40H omswflm ma NH m d I I ‘9 an XSPUI DlOIdfiIOJ fl uI DIOIdAIOJ L XQD in different seaments of the mitotic cycle may be summariz- ed as follows: 1) Cells in the fourth, iiftl., and sixth hour sebments of interehase are, as COMyareQ to control, a) usually delayed two hours before enttrinb into division and b) generally less dispersed and therefore better synchronized. 2) Cells in late interphase (eibtth and ninth hour sebments) divide less synchronously as compared with the control while, ()3 cells of the filth hour segment divide earlier in the second division than either the control, the fourth hOur treated or the sixth hOur treated popu- lations. DNP has been classified for quite some time as an oxidation-phosphorylation uncoupler, that is, when livinD tissue is eXposed to uhr, oxygen consumption usually in- creaSes markedly while the production of phosthorylated com- pounds show no parallel increase (Loomis and Lipman, 1945). LNP mibht be called the classical UUCOUylGP althoubh there are many other similarly actihb compounds. One of these is 3, 4-dichlor0phenol (LOP). DC? has been shown by Krahl and Clowes (ldb7) to affect sea urchin ebbs in a Way similar to LNP (Clowes and firahl, 1957). boP has also beenreeorted as having the same cytolobical effects as UN? only at 5reater concentrations (Muhling at 31, 1960). Furthermore, DC? has been shown to exhibit the same physiolohical effects as th on plant tissue (daur and peevers, late). Therefore, it seemed lobiCal to determine whether or not DC? would have the same eiiknyts as oNr on cells in different sebments of the mitotic cycle. The results 01 an experiment where cells were treated at either 4, b, or 6 hours after marxinb are shown in Figures 11A, llb, and 116. It is evident that the experiment with DCP corroborates tlose of DEP and that the summary of the effects of uNP also apply to GP. VerifiCation of the ewe results by ed? stimulated some speculation as to the nature or cause of the acceleration of the mitotic cycle. ode to the interest shown in the biochemiCal activity of DB? a larbe number of reports have been published dealinfi with this subject. In COUSldePhlé these, it appears that perhaps the most general effect of DNP in gizg is the activation of the enzyme adenosine triphOSphatase (ATPase) (Cooper and Lehninber, 1357; ten- hiall, 1960; fallman et 31, lddO; Simon , 15:5). This ac- tiVation could result in an increase in the intracellular concentration of ADP (adenosine diphosphate) and k1 (in- organic phosphate) since ATPase catalyzes the following reaction: ATPaSG ATP -------- > ADP Pi The characteristics of the DNP-stimulated aTPase reaction have been reasonably well described by rullman 33 El’ iaeo). Further, since the;rate of anaerobic slycolysis has been reported to be dependent on the concentration of auP OHH enjmfia ma 1 q \xJa” mecca an am vm Om doumoae A7..I.II¢ Hoppzoo ATIIIIII; 2 m xenuI prordfltoq .onfloagoaoo spas ncfixnwe Longs Aoaa opdmflzv made; mecca .Acaa opsmflav majog m .Amaa endsfiav mason e um mom 2 enoa x n.v hue: H usofiamohg oaJEME ma w Mo pomggm 0:6 om nan assess aaa assess mhzow wm Wm ON 0H ma tlm WHJOA NM om vm ON ma NHerw 141- u 1 1-: fl 4 1 4 1 u . u 114 q 1|I~ W all s \ v TL ‘ ‘ W ~ 4 ”M a K 3 \ \Lm T. . MW 0 0 T m .12”: I a s . a J m . m c .. . N9 /\ , \ la, X .m X :. a . Chance and bees, late; ummlot and nos, lace; hess and Chance, lael; Johnson, ludl; hardy and raras, laud; Lehninber, 1357), it is quite possible that the accelerated cycle may be the result of a temporary stimulation of the hmbden-heyerhof pathWay due to a momentary increase in in- tracellular an? and ll. If this were so, then a combination of an anaerobic glycolytic inhibitor and th should show a different reSponse than either the blycolytic inhibitor or DNP used sinbly, bibures lbn, lab, 120, and lay and 125 show the results of such an experiment. In this case the inhibitor fluoride was used. The use of fluoride was based on its ability to form a complex with magnesiumvvhich is a necessary cofactor for enolase. Figure 12a represents graphically the effect of a lb minute treatment at the fifth hOtr with 0.01 M fluoride. The data indiCate that the fluoride had little influence on the tetraploid pOpulation and its appearance in division II. fibure lab however, shows that a similar treatment with 0.05 M fluoride does delay the appearance of the marked cells in division I but does not decrease the cycle time be- tween divisions I and II. libure 120 supports previous obserVations in that a lb minute treatment at the fifth hour with UN? both delays division I and accelerates the cycle leadin5 to division II. li5ure 12D shows the results obtained by a combined treatment with 4.32 i 10‘5 M DNP and 0.01 M fluoride at the fifth hour. The combiued treatment obviously delayed sandman: A.ll|l.v ”Hoapcoo Aolulle .ecfloanoaoo Spa; 3:353... .3th .35: S; :H ogp on 73H ease H: ocwuosam Hod 9.13 .83. a mloa 4 OJ. 23 .AQJH endoflav a3 ; )IOH 4 3% Ana sauna: 03:05.: a no. :4NH oasnaj oadanosaw 4 Ho. 0 nods anodes. cap epic as ea 1 Mo #00990 2:. Qua Poznan omH unseat made: mm vn ON 0H NH 3 muse: mm «up. On. ma NH m - . a 4 fl 11 u 1 .l a - vd M n m. .L .A a m U m o o t. I. P D. I 0H” u v D. 9 m .1... J AFN ama 8%: <3 3%: mono; aw em om ma NH m maze: an em. ON ma w. a .d < - In I Cd 0 0L o I T. K F. n a o m. o T. TL- 0. D I (“HI u U D 0., .. . an m .8 w Adm gem L L0 the pOpdlatitnitdni Caused an increase in synchrony. More important however, the acceleratinb effect of th appears to he somewhat dampened by the fluoride. n comparison be- tween Pibures lac and lab certainly succcsts that an in- hibition has occurred. The inhibitory effect of fluoride is rather pronounced when th and 0.0b h fluoride are combined. libure lea shows a very breat delay in the first division of the marked population. This lenbthy delay is probably the result of both UN? and the fluoride, since our itself delays and, as figure lbh indicates, the 0.0b h fluoride also proouces a delayinb effect. The obserVations made on the fifth hour segment of interphase with these eXperiments may be summarized as: l) 0.01 M fluoride appears to have no effect on the pOpulation in any way, 2) 0.05 M fluoride preduces a delay in the appearance of the marked cells in division I out does not de- crease the duration of the cycle between divisions I and II, b) fluoride when combined with hNP tends to prevent the decrease in cycle time between the first and second division after treatment. These observations support the hypothesis that con- ditions favorih5 anaerooic blycolysis at the fifth hour segment tend to accelerate the second mitotic cycle follow- ing treatment. There is, however, some question concerniné poodbaH.Ao.....& “Hoapzoo AQIIIIIIJ .odflofigoaoo Sow; mcflxase Longs ado: Loufim 9:9 as ooaaosaw 2 no.0 msad tag 2 inoa & fi.v Qua; poosonoAp ooscHE ma a 90 became oza .dma dugout i mason Ofl wN 0N ¢N NN ON nfi 0H vH NH OH Q - IV - >1 1 11 1 J 1 m. 0 06 a 0 La L0 in [NH 10H IQH lam em. xspuI DIOIdKIOJ PC.) the specificity of the inhibition by fluoride. rullman et al, (1960) have shown that potassium fluoride will in— hibit the activation of aTPase by LAP to a deéree. This inhibition occurs because magnesium is a cofactor in the reaction Catalyzed by nTrase. The effect of fluoride therefore, may not be the result of enolase inhibition but rather the result of the inhibition of aTPase aCLiVation by DNP. hence some doubt remained as to whether or not an- aerobic glycolysis Was favored by a short time treatment with th. It seemed quite possible that both aTrase ac- tivation and anaerobic blycolysis were effected by the fluoride. because of this uncertainty another series of eXperi- ments involvins the use of potassium Cyanide Was desiéned. Potassium cyanide was chosen beCause its use in a short time treatment would tend to increase the rate of anaerobic glycolysis. This would presumably come about by increasing the amount of intracellular abP and Pi° As pointed out earlier, the increased availability of ADP and Pi should increase the rate of the hmbden-Meyerhof pathway. Botassium cyanide, moreover, differs greatly from tar in the mode of inhibition. Cyanide does not activate aTrase nor does it increase oxygen consumption in the treated tissue. cyanide does however, inhibit oxyben consumption and prevents the operation of the cytochrome system by combining with enzymes having metalloporphyrin units. The first series of experiments with cyanide Was de- sibneu to find out if it had the same effect as uh? on cells that were in late interphase. The concentration used in these CXperiments Was 4 h lO-b m. This concentration Was shown by Lichenberber and Tiimann (1957) t» inhibit rCSpira- tion in the pea. ribtre in shows that a l5 minute treatment at 7, U, or J hours delays the cells a little bat does not approach the degree of delay preduced by UNP. Figure 14 a indicates that a similar treatment with cyanide at the filth hour does not improve the synchrony of the population even thoabh it is delayed. It is important however, to notice that the second appearance of the pogu- lation occurs before that of the control. Therefore, a fifth hour treatment with cyanide procuces the same result as DNP with regard to the time of onset of the second di- vision after treatment (Figure 14 n). hhen a comparison is made between libures 14 a and l4 b, the difference and the similarity of these two chemiCals are well illustrated. It is also interesting that when the pOpulation was treated at the fifth hour by UhP and cyanide combined, the effect pro- duced was neither complementary nor antaéonistic (bibare 14 C). The next series of eXperiments involvinb potassium cyanide was desibneo to detect differences, if they existed, between the fourth, filth, and sixth hour seéments with re- spect to the preduction of an accelerated second cycle. The duration of treatment and the concen ration of Cyanide used in these experiments were the same as those mentioned earli- .Hoppsou Aollllle .20 m 3Q . . bSaxasE LWNHM Aom baoaaeiaom ego Ho Coawfl>wo pmpflm ozn no QCHOHgOHOO Qua; . . -. . O o o. 00v white; m. USA. a. Aoll. o IIOV mpHSOL ® a A till“; .WLJOQ a we moacmho 3 I IOH K v Spas pCeEpsoLp QQJQHE 0H a mo pooM%m eLB .mH ebzmaa \J J maze; we on ma 2 3 ma 2 m. . q I. q 1 ~ J O o 7d O .m. m_d 4A.”; Th _.. O I. Di Q CHI U D. 3 max ma Hm ¢L sew copmoLB ?.l.ln¢ Hoapdoa Aflllllv .coaosasaom oaoaashoop use no GOHmH>Ho ecooem dam umaaa exp :0 ecfloanoaoo new; finaxase Looms Leon guwwm mzp as ona omdmflav mag a n-0a , n.a mafia measaaa 2 0-0a a a sea . uaa magmaaa aaa a 3-0a a n.a .sza oadbflav ooflsemo a nod A e Leas it useEosotp epssfle ma 5 .o became one sea oasoaa mascaa mm wt ON ma xepuI DIOIdKIOd sea cassava ma a L! made; mm em om. ma L «an wasmaa shawl. am am on ma ma an 1‘11. J G d x s a \\. \ [‘0 r4 "Q g 0) DUI QIOIdKI°& X9 .hm H m .0 ca xepul ptoIdKIOJ 1‘ 01 Lb er. by comparinb the beheral pattern of the Curves shown in figures 15 a, lb b, and lb C it is evident that treatment with cyanide at early interphase does not produce a delay in the appearance of the tetraploid population and only the fifth hour treatment brinbs about an acceleration of the second cycle following treatment. Furthermore, ribure lb a shows that the accelerated second cycle can be prevented somewhat by 0.01 M fluoride. These results suggest further that conditions resulting in an increase of anaerobic blycolysis durinb the fifth hour Segment of the mitotic cycle results in a decrease of the cycle time of the second division following treatment. A summary of the obserVations made on the cyanide ex- periments is outlined below. 1) Cyanide does not improve or decrease the synchrony of pepulations treated in either late or early interphase. 2) The delay produced by cyanide appears to be the result of a general effect on all cells in the pOpulation. 5) Only the fifth hour segment of interphase responds to cyanide treatment in the production of an accel— erated second cycle. a) 0.01 M fluoride prevented the acceleration of the ‘ -.. " ,v'; ~ 18 second cycle preduced by cyanide treatment at tn fifth hour. ceaeehm.fitu.l.ll¢ “Hoausoo Afillllllv .eeaaaasQOQ afloadeeaua aha do coaaa>aa season can ameaa azaaxc ecflommoHoo 39H: acaxtsa Longs mezox m as Cqu meataav moaaozdw a H0.0 mSHu U 1. o o a v do acn;o a muOH A v go one uwH epswaav mass; @ .ADJH madmagv dado: o .Aqu OnHZ;.HHV WeHSOq . \m . r a .. u U . A v we evade o 2 muOH x w Sow; psoEpmoLp opdcfie nH m we poemme eAB . 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