Cancer Research

CANCER RESEARCH

VOLUME12 DECEMBER 1952 NUMBER12

Some Investigations on Cell Behavior under Various Conditions: A Review*

HANSLETTRÃ‰

(Institut fÃ¼rexperimentelle Krebsforschung der UniversitÃ¤t, Heidelberg, Germany)

Some of the basic problems of cancer research Tissue culture (%4)-â€”Several cultures of arise in the field which we call cytobiochemistry. chicken mesenchymal or heart fibroblasts, 48 Besides fundamental research on the chemistry of hours old, were divided into two portions. One half cell behavior, we have been especially interested was explanted as a control culture in a medium in the search for substances with tumor-inhibiting consisting of 1 drop of chicken plasma, 1 drop of properties. This review is a survey of work on embryonic juice, and 1 drop of saline solution. The mitotic poisons, their antagonists and synergists, second part, serving as the experimental culture, the chemistry of cell division, the relation between was explanted in a medium containing the solution cell metabolism, growth, and division, and the be of the substance to be investigated instead of the havior of components of tumor cells in transplan pure saline solution. The final concentration of tation. the added substance was one-third that of the 1. MITOTICPOISONS added solution and is expressed in /ig/ml. At the In 1937, Dustin (11) described the ability of end of 24 hours, control and experimental cultures colchicine to inhibit mitosis of cells (normal and were compared. The effect of a mitotic poison is so malignant) in vivo (12,13). Colchicine is one of the drastic that the effect can be observed qualitative compounds that Dustin called "poisons caryo- ly in the living culture (Figs. 1 and 2). For quanti clasiques." In 1939, Ludford (82) described a simi tative estimation, the cultures were stained and lar action by several compounds on cell division the numbers of mitoses counted and morpho in vitro; he called these substances "mitotic poi logical changes recorded. To investigate effects on sons." We found that not all the substances which the cell spindle, the staining method of Ehrlich- Dustin called "poisons caryoclasiques" produced Biondi was used, and the stained preparation was an effect on cells in vitroâ€”i.e., a direct action on observed under the phase contrast microscope (24) the cell in division. Thus, we propose to call a (Fig. 3). In the case of colchicine, 0.01 jig/ml can substance a "mitotic poison" only if its direct be detected by its action on dividing fibroblasts. action on the cell has been proved. Examples of Since 1939, more than 1,000 chemical compounds such substances are colchicine, acriflavine, podo- have been investigated by this method, involving phyllotoxin, auramine, and others. about 350,000 cultures of fibroblasts (reviewed by Any type of dividing cell of animals or plants LettrÃ©,81, 35). We used this method to estimate would be suitable for testing the effect of a sub the distribution of colchicine in the body; extracts stance on mitosis. We used two methods: tissue of different organs were tested for their effect on culture (including fibroblasts, carcinoma, normal tissue cultures. By this methodâ€”comparable in its epithelium and other tissues) and the mouse sensitivity to the method of isotopically labeled ascites tumor. compoundsâ€”we found that colchicine is excreted * Based on talks presented at the Gordon Research Confer chiefly by the liver via the bile into the intestine; ence, AAAS, on August 27,1951, and at the University of Wis there is no urinary excretion. Even when toxic consin on September 20, 1051. dosages were administered, the brain contained Received for publication April 30, 1952. no amount detectable by this method (75). In 847

unpublished experiments with Roeder (GÃ¶ttin vival time of the control group and that of the gen), we found that colchicine derivatives intro treated group gives a further indication of the duced by means of iontophoresis into the brain of activity of a substance. In 1934 Collier and Jahn guinea pigs had no acute toxic effect; the colchicine (7) mentioned the mitotic figures in the dividing derivative could be detected in the liver 10 tumor cells and called this tumor an excellent ob minutes after the iontophoresis. ject for cytological experiments. Brodersen (4) de Motion pictures are an important aid in study scribed a morphological method for investigating ing the action of mitotic poisons on cellular be the mode of action of a substance. Smears of ascites, havior. Films of our studies are available to uni taken at different times after the injection of a versities (33, 34). substance, were stained and the number of mitoses and morphological changes recorded. Brodersen 9 thus was able to differentiate between the action of 38 colchicine, acriflavine, and x-rays (Chart 2). We 36 'Sf use this method routinely to compare chemical compounds. The effect of N-methylcolchicamide Sf (NMC), a more effective derivative of colchicine, sa was demonstrated by this method (Fig. 4) (62). Â¿8 By fractionating the dosage of NMC, we could 26

it 100 a to it 80 Â« n 60 u Z # t t 10 Ig 1t 1f n 20 22 2t 2S 2S 30 days CHART1.â€”Weightcurves of mice with ascites tumor. 40 7â€”At A, 0.2 ml. tumor ascites inoculated intraperitoneally. 77â€”Thesame and at C, 10 Â¡ig.colchicine daily, as indi cated. 777â€”Notumor inoculated; at C, 10 /ig. colchicine daily, as 20 indicated. 7 Vâ€”Notumor inoculated ; at C, 20 pg. colchicine daily, as indicated. JI lililÃ I I i tâ€”Death of mice. 0 4 8 12 16 20 24 28 32 36 40 44 48 52 CHART2.â€”Effects of different agents on the number of The mouse ascites tumor.â€”In 1932 Loewenthal mitoses of the mouse ascites tumor. and Jahn (81) described a subline of the Ehrlich 7â€”Colchicine. 77â€”Acriflavine. mouse carcinoma. By intraperitoneal injection of 777â€”X-Rays. free cells of this tumor they succeeded in obtaining Ordinate: mitoses per 1,000 cells. an ascites containing free tumor cells. Since 1940 Abscissa: hours after treatment. we have used this ascites tumor for studying the influence of exogenous factors on its growth (25, show, for instance, that five injections of 3 ^g. of 26, 36). The production of ascites, together with NMC, given every 24 hours, are much more effec the increase in the number of tumor cells, drasti tive than a single injection of 15 jug- (LettrÃ©and cally changes the weight curve of the mouse. The Bergdolt [55]). increase in weight may be 20-30 gm. Thus, the Following repeated injections of small doses of weight curve of the animal after the injection of the NMC and repeated transplantations of tumors tumor cells is a good indicator of tumor growth. treated in this way, a subline of the mouse ascites Following the injection of mitotic poisons, the tu tumor was obtained which was resistant to this mor growth was inhibited, and the difference be agent. We consider this resistant kind of tumor to tween the weight curves of the experimental be the result of a spontaneous mutation. This animals and the control group can easily be ob subline is also resistant to colchicine (61). Al served (Chart 1). A comparison between the sur- though cells of this resistant form divide by

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1952 American Association for Cancer Research. LETTRÃ‰â€”CytochemicalStudiesof Cell Behaviorâ€”AReview 849 mitosis, division cannot be influenced by colchi- removes the acetyl group. The resulting compound, cine or NMC. This finding is important for the trimethylcolchiceinic acid, has no activity as a theory of mitosis. It is important, too, for an un mitotic poison (cf. Brues and Cohen [5]). Thus, derstanding of the action of colchicine on the we might conclude that the acetyl group is in normal form of the ascites tumor. The objection dispensable for activity. However, if we transform has been raised that the inhibition of tumor this compound into its methyl ether, desacetylcol growth was only secondary to a primary effect on chicine, we get a substance of almost the same the host. In investigations with the resistant form activity as colchicine, although it does not contain the hosts receive the same injury from the sub the acetyl group. Trimethylcolchiceinic acid is an stance as in the case of the sensitive form. There amphoteric system, and this seems to be the reason fore, the inhibition of the sensitive form is actually for its inactivity. These results, published in 1942 a direct effect on the tumor cell and not a sec (29), are in agreement with the findings of Ullyot ondary phenomenon. and his associates (14, 17). Windaus (93) trans Colchicine and derivatives.â€”The formula of formed colchiceine to N-acetyliodocolchinol and colchicine (Table 1, I) proposed by Dewar (9, 10) acetylcolchinol; both substances are effective anti-

TABLE 1 TABLE 2 COLCHICINEANDDERIVATIVES ANTIMITOTICACTIVITYOFCOLCHICINE DERIVATIVESONCHICKENHEART FlBROBLASTS Level allowing NH activity Derivative (fig/ml) colchicine 0.01 isocolchicine 0.4 colchiceine ethyl ether 0.04 isocolchiceine ethyl ether 9 colchiceine propyl ether (mixture) 0.8 colchiceine butyl ether (mixture) 2.5 FORMULAI colchiceine 4.5 Ri-CO-CH,-CO-CH,-H-H-CO-CH,-CO-CH,-CO-CH,-CO-CH,-CO-CH,-CO-CH,-CO-CH,R>-OCH,-OH-OH-OCH3-OC2H5-OC,H,-OCiH,-NH2-NH-CHs-NH-C2H6-N(CH,),desacetylcolchicine 0.05 colchicine trimethylcolchicinic acid 100 no activity colchiceine colchicinic acid 100 no activity trimethylcolchiceinic acid colchicamide 0.01 desacetylcolchicine N-methylcolchicamide 0.0025 O-ethylcolchicine N-ethylcolchicamide 0.003 O-propylcolchicine N-propylcolchicamide 0.08 O-butylcolchicine N-butylcolchicamide 0.9 colchicamide N-dimethylcolchicamide 0.005 N-methylcolchicamide N-methyl,N-propylcolchicamide 0.5 N-ethylcolchicamide N-benzylcolchicamide 3 N-dimethylcolchicamide N-acetyliodocplchinol 0.4 N-acetylcolchinol 0.6 FORMULAII colchinol 0.1 -CO-CH, -OH N-acetylcolchinol N-methylcolchinol methyl ether 4 -CO-CH, -OCH, N-acetylcolchinol methyl ether N-dimethylcolchinol methyl ether 5 -H -OH colchinol hexahydrocolchicine 0.5 oxycolchicine 10 in 1945 now seems to be well established. Colchi trimethoxyhomonaphthide 80 no activity cine, an alkaloid from Colchicum autumnale, con N-acetylcoIchinic acid anhydride 60 no activity tains in its molecule three rings: one aromatic ring with three methoxyl groups, one seven-mem- mitotic agents. Colchinol (Table 1, II), obtained bered ring with an acetylated primary amino group, by hydrolysis of N-acetylcolchinol, is the simplest and one tropolone ring with a methoxyl group. degradation product of colchicine that has activity. Since 1940, we have been investigating degrada If the tropolone ring is destroyed by oxidation tion products and derivatives of colchicine to (N-acetylcolchinic acid anhydride) or if the nitro clarify the relationship between chemical con gen is removed from N-acetylcolchinol methyl stitution and activity as mitotic poison (58) ether to desaminocolchinol methyl ether or to the (Tables 1 and 2). The hydrolysis of colchicine so-called trimethoxyhomonaphthide of Windaus, leads to colchiceine, the methyl group (R2 in there is a complete loss of activity. Tropolone and Table 1) being split off. Colchiceine is still a its methyl ether do not show any antimitotic ac mitotic poison, but quantitatively its activity is tivity (71). only -fija of that of colchicine. Further hydrolysis By remethylation of colchiceine with diazo-

methane, two substances result: colchicine and an structure to colchicine. At this time, the Windaus isomerie substance, isocolchicine (Sorkin [88]). formula of colchicine (93) was still accepted. Isocolchicine is a derivative of an isomerie form in Cook and Engel (8) made open-ring analogs of the tropolone ring. The activity of isocolchicine is compounds corresponding to this suggested for â€¢fathat of colchicine. The ethyl ether of colchi- mula of colchicine. In applying their principles to ceine, corresponding to colchicine, has Â£the activ the Windaus formula, LettrÃ©and Fernholz synthe ity of colchicine, but the ethyl ether corresponding sized 4'-methoxy-a,/3-diphenylethylamine (4'- to isocolchicine only 1UI6<)(59). methoxy-stilbylamine) (Table 3) (58) and found it All these compounds are less effective than active on fibroblasts at 4 ^g/ml. a-Phenyl-7(p- colchicine. The reaction of colchicine with am methoxyphenyl)-propy lamine (compound IV), monia produces colchicamide, comparable in its CH2- CH2- C-NH2 TABLE 3 SOMEEXAMPLESOFTHE RELATION BETWEENSTRUCTUREANDANTI- MITOTICACTIVITYINTHE STIL-

BYLAMINESERIES OCH, 3' 2' IV. a-Phenyl-7-(p-methoxyphenyl) propylamine CH2-C-NH2 analogous to the new colchicine formula, did not show any antimitotic activity. Therefore, we thought that the Windaus formula for colchicine was correct with respect to the size of the B-ring III. 0,0-Diphenylethylamine ("Stilbylamine") and the position of the nitrogen. We investigated a great number of derivatives Level showing of a,/3-diphenylethylamine ("stilbylamine") and activity Substituents (eg/ml) found a number of active compounds (Table 3) unsubstituted none (56, 57, 58). The 4'-ethoxy derivative is the most 4'-methoxy- 4-5 active of these synthetic compounds; it was sepa 3'-methoxy- 20 2'-methoxy- none rated into its optically active forms (77). Only 4'-ethoxy- 0.4 the isomer with ( â€”)-rotation proved to be effec 4'-propoxy- 0.8 tive; the (+) -isomer has no or only yfoy of the 4'-butoxy- 1.8 S',4'-dimethoxy- none activity of the ( â€”)-isomer. Hence, its activity as a 4,4'-dimethoxy- mitotic poison is stereochemically specific. 4-methoxy- 3',4'-methylendioxy- 6 The simplest synthetic compound with an anti 3',4'-dimethylendioxy- 4 mitotic activity on fibroblasts is 1-aminoacenaph- S',4'-tetramethylendioxy- none 3',4'-hexamethylendioxy- thene (V), CizHnN. However, compared to S',4',5'-trimethoxy- 4'-methyl- 10 3',4'-trimethyIen- 10 S',4'-tetrametnylen- 10 2',3'-benz- 3',4'-benz- S,4,3',4'-tetraraethoxy- 3,4,3',4'-(methylen-dioxy)- V. 1-Aminoacenaphthene activity on fibroblasts to colchicine. A great num ber of substituted colchicamides were obtained by colchicine, CuuHziAsN, its activity amounts to the analogous reaction of colchicine with primary onlyTOTOâ„¢(79)- and secondary amines (28). The activity of N- Alkaloids. â€”It seemed of interest to determine methylcolchicamide (and the N-ethyl and N- whether other alkaloids would have an effect on dimethyl derivatives) is greater than that of cell division similar to that of colchicine. We tested colchicine. The derivatives bearing substituents 83 alkaloids and derivatives for their action on with longer carbon chains on the nitrogen atom fibroblasts (50, 51). These alkaloids are listed in have less activity (LettrÃ©[36, 47, 67]). two groups (Tables 4 and 5) : the first group has Synthetic analogs of colchicine.â€”In 1940, we no chemical relation to the stilbylamine group, started to synthesize substances similar in their the second group are alkaloids which contain the

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1952 American Association for Cancer Research. LETTRÃ‰â€”CytochemicalStudiesof Cell Behaviorâ€”AReview 851 stilbylamine group. Among the first group there an extraordinarily strong effect, even surpassing was no mitotic poison; but in the second group we that of colchicine on these eggs (Table 6). We may found several acting as mitotic poisons : narcotine, visualize the mechanism of action of these quiÃ±ones gnoscopine, chelidonine, homochelidonine, meth- to be a reaction with SH-groups, since p-benzoqui- oxychelidonine, and protopine. It is striking that TABLE 6 TABLE 4 ANTIMITOTICACTIVITYOFSOMEQUI ALKALOIDStyraminehordenineephedrinemescalinehygrinenicotineconiinelobelinepiperineatropinehyoscyaminescopolamineecgoninecocainepelletierineNOTSTILBYLAMINE(NotRELATEDTO Ã‘ONES,COMPAREDTOTHATOF mitoticpoisons)lupininelupinidinecinchoninecinchonidinequininequinidinecinchoteninepellotineanhaloninecarneginecotarnineyohimbineaspidosperminepiloacarpinephysostigmineeseridineaconitinestrychninebrucineemetinecephaelineergotaminelycopodinelycoctonineagaricinearicinegelsemininetaxineveratrinesolanine, STILBESTROLANDCOLCHICINE Minimal active Antimitotic dose (for substance Tubifex eggs) stilbestrol 1:300,000 colchicine 1:30,000 benzoquinone 1:2,000,000 naphthoquinone 1:900,000 phenanthrenequinone 1:30,000,000

none can add cysteine. Thus, the effect of quiÃ±ones would be the consequence of SH-group blocking. The investigation on the effect of epinephrine on tissue cultures resulted from a study of the mitotic poisons of the stilbylamine type which TABLE 5 contain the sympathicomimetic phenylethylamine ALKALOIDSRELATEDTO STILBYLAMINE group. Numerous derivatives of this group, such (Mitotic poisons in italics) as mescaline, tyramine, hordenine and others, I. aporphine group: oxyacanthine proved to be inactive (49). Only epinephrine apomorphine rhoeadine showed a detectable activity with a dose of 100 morphothebaine d-tubocurarine corytuberine III. berberine group: Mg/ml. After the addition of epinephrine, the corydine berberine tissue culture medium became red-brown, so that bulbocapnine tetrahydroberberine we may assume that an oxidative product was boldine 16,17-dihy drodesoxy ber glaucine berine formed which had antimitotic activity. Thus, we lauro tetanine palmatine can define epinephrine as a promitotic poison. laurotetanine methyl ether tetrahydropalmatine When at the same time either vitamin C or gluta- N-acetyllaurotetanine jatrorrhizine II. papaverine group: tetrahydrojatrorrhizine thione was added, oxidation of epinephrine was in papaverine tetrahydrocoptisine hibited and, in consequence, its effect on mitosis. laÃºdanosme IV. chelidonine group: pavine chelidonine For a long time we have searched for an oxidative Â¡audanine homochelidonine product which in its constitution is related to laudanosoline methoxychelidonine stilbylamine. In 1947 however we found that pure narcotine chelerythrine gnoscopine sanguinarine adrenochrome (VI) has this effect (74). According narceine V. cryptopine group: hydrastine protopine morphine cryptopine : -OH codeine the active alkaloids contain the stilbylamine group. It was mentioned before that this structural element seems to be essential for antimitotic VI. Adrenochrome activity in the synthetic compounds. The type of substitution is of importance for activity, and this to this, we suggest that the active product of may explain why not all alkaloids with the epinephrine is effective on cell division through stilbylamine group are active. its quinonoid character. QuiÃ±ones.â€”Theinhibiting effect of quiÃ±ones on Organometallic compounds.â€”The discovery of cell division was discovered by E. F. Lehmann the effect of mitotic poisoning by organometallic (23). For his experiments he used eggs of Tubifex compounds was stimulated by A. Klages (21). He (a species of ringworm which lays eggs during the observed that organic compounds of Hg which whole year and thus is on hand for experiments were being tested for antimycotic activity induced at any time). Lehmann found that benzoquinone polyploidy in the treated seeds. At the beginning naphthoquinone and phenanthrenequinone had of 1943, we tested the effect of organic compounds

of mercury on chicken heart fibroblasta and ob monoaminoacridines. Among the tested 3-, 4-, and served an inhibiting effect on mitosis. Many 9-aminoacridines, only the 3- and 9-derivatives (X)

compounds of the type Hg-R-X were effective, R NH. being an aliphatic or aromatic organic radical and X, an aniÃ³n.Inorganic salts of Hg such as HgCl2 and Hg(CN)j, as well as compounds of Hg com pletely substituted with organic radicals like Hg- diphenyl, proved to be ineffective. Similar be X. 9-Aminoacridine havior has been observed with other metals, e.g., lead, bismuth, tin, arsenic, and antimony, which proved to be effective, the 4-amino derivative also show antimitotic activity only when in mixed showing no effect. Quantitatively, the activity was organic-inorganic compounds (31, 32, 35). considerably lessened. For chicken heart fibro Mitotic poisons of the acriflavine type.â€”Acrifla- blasts 100 /Â¿g/mlwere needed, whereas proflavine vine (VII) is the methochloride of 3,6-diamino- was effective at 2 /Â¿g/ml.We found simpler com pounds related to 9-aminoacridine which were also effective. The condensed aromatic ring sys tem may be omitted without loss of activity. 4-Amino-quinoline (XI) and 4-aminopyridine

VII. Acriflavine acridine. Its effect can be demonstrated on both fibroblasts (6) and mouse ascites tumor (27). Our first investigations dealt with the question which XI. 4-Aminoquinoline of the substituents in the molecule are necessary for the effect (31, 32, 35). Proflavine (VIII), or (XII) showed an inhibiting effect on cell division

NH.

VIII. Proflavine XII 4-Aminopyridine 3,6-diaminoacridine, had the same effect, qualita as well. tively as well as quantitatively. Acridine and 4-Aminopyridine was the simplest active mole acridine methochloride, however, were ineffective. cule which we obtained in the acriflavine series. Thus, the amino groups in the acridine skeleton According to our former investigations pyridine appeared to be indispensable for activity. We then itself showed no inhibiting effect on cell division. tested whether a molecule similar in structure to A comparison of the three isomerie aminopyridines proflavine and with the same distance between the proved that the 2- (XIII) and 4-aminopyridines amino groups would show the same effect. 2,7-Diaminoanthracene (IX) is such a compound.

NH, XIII. 2-Aminopyridine

IX. 2,7-Diaminoanthracene were effective, whereas even high dosages of 3- aminopyridine were ineffective. The inactive Even in high dosages it showed no inhibitory effect 3-aminopyridine differed chemically from the on cell division, nor did 2,7-diaminocarbazole. other two isomerie products, inasmuch as a Likewise, "open" molecules built up in analogy to tautomerie form of it does not exist, whereas the proflavine, as such p-diaminodiphenylmethene effective isomers were able to form products of the and m-diaminodiphenylamine, were ineffective. damine type. Likewise, the active aminoacridines Hence, analogy to the form of the molecule of pro and aminoquinolines could form tautomerie flavine was not essential for activity. We obtained products, while the inactive members of these further information from the investigations on series could not. The inactivity of aromatic amines

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1952 American Association for Cancer Research. LETTRÃ‰â€”CytochemicalStudiesof Cell Behaviorâ€”AReview 853 of similar structure bore out this conclusion. the discovery of factors able to act in an an Thus, we may summarize the results as follows: tagonistic or synergistic way. aminoacridines, aminoquinolines, and aminopyri- From the morphological point of view, acrifla dines produce an inhibiting effect on cell division vine represents the classical type of a chromosomal provided that they may form tautomerie products poison, according to Bauch (2) and to Bucher (6). of the diimine type. It produces sticking and clumping (pyknosis) of If the mitotic poisons of the acriflavine type chromosomes. Chemically, the effect of acriflavine act as antagonists of the purines and pyrimidines is attributable to the formation of a complex with in the nucleic acids, all chemical analogs of purines the nucleic acids. We were able to counteract the and pyrimidines ought to be mitotic poisons of the effect of acriflavine on fibroblasts in vitro by acriflavine type. Experiments do not confirm this adding nucleic acid to the culture medium (either hypothesis. However, we are able to increase the ribo- or desoxyribonucleic acid) (64). These results activity of the basic types of aminopyridines by are similar to those of Mcllwain (83) with substitution, producing a closer analogy to the bacteria. Brodersen (4) showed that the injection natural pyrimidines. 6-Methoxy-2-aminopyridine, of acriflavine decreased the number of mitoses in for instance, is more effective than 2-aminopyri- mouse ascites tumor, thus demonstrating its influ dine itself, possibly since the methoxy derivative ence on nucleic acid metabolism. The reaction is more similar to cytosine. However, a number of with the chromosomes is easily shown morpho synthetic analogs, e.g., aminothiazoles, amino- logically, but a reaction with the ribonucleic acid quinazolines, and aminotriazines, produced no of the mitochondria can be proved just as easily: effect on chicken heart fibroblasts. enzymatic reactions of the mitochondria can be Hormones.â€”As mentioned before, epinephrine inhibited by acriflavine. It seems important that acts as an antimitotic agent after its transforma during the time of the minimum of mitosis in the tion into adrenochrome. We tested the effect of a ascites tumor we find a maximum of amitotic great number of hormones on cell division of fibro divisions (78). We can summarize the action of blasts. Only estradici, in the form of the sodium acriflavine as follows: the nucleo tides and the salt of its phosphoric ester, had an inhibitory ef nucleic acids of the cell (both those of the nucleus fect at a dosage of 40 ^g/ml. Stilbestrol also pro and those of the cytoplasm) react with acriflavine, duced a very pronounced effect (30). Since hor thus inducing a disturbance of either the polymer mones are regulators of metabolic processes, we ization or the reactions of the nucleic acids. The must assume that the processes of cell division are morphological aspect is modified by the dosage also regulated by hormones and that hormonal and by the stage of the cell at the moment of re specificity exists in the regulation of different action. types of tissues. The antimitotic action of organometallic com Although adrenochrome inhibits mitosis of pounds could be counteracted by the addition of fibroblasts, it has no effect on carcinoma cells in cysteine or other compounds containing SH- vitro. This forms the basis of our hypothesis (29, groups (65). Thus, we consider their mode of action to be a reaction with SH-groups of cell 49) that malignancy results from a type of muta tion of a cell, as a result of which hormones regu components which are functionally important during mitosis. There are many enzymes the activ lating the normal type are no longer effective ity of which depends on their number of free SH- against the malignant type. Although the regula groups and which are important for cell metabo tors are present, they are ineffective because the lism but not directly for cell division. According to cell has changed. The alterations of cell metabo L. Rapkine (85), a maximum of free SH-groups lism alone would not explain the capacity of a occurs just before cell division. Thus, at that mo malignant cell for unlimited growth under condi ment the organometallic compounds can react tions where a normal cell is not able to grow. There with certain SH-compounds important for cell must exist a disturbance of the regulating system division. Godeaux (15) was the first to show that between cell and host. the contractibility of actomyosin is inhibited by poisoning SH-groups; the antimitotic activity ÃŽ.ANTAGONISTSANDSTNEHQISTSOFMITOTICPOISONS thus may be interpreted as the inhibition of the For the most important types of mitotic poisons contractibility of cell components indispensable we are able to determine the point of attack in for mitosis. side the cell during mitosis. The determination of The mode of action of colchicine was eluci the mechanism of action is based on (a) the dated by synergistic compounds rather than by morphological picture of the treated cell and (b) antagonists. We found a great number of sub-

stances which increase the activity of colchicine on The behavior of the steroid hormones is similar fibroblasts in tissue culture (44, 45, 63, 66-73). to that of the alkaloids. Most steroid hormones These substances belong to different chemical have a synergistic effect; again, there are two groups. These substances can be divided into two types: one increasing the activity of a threshold groups : the members of the first group only show dosage of colchicine, the other able to activate an a synergistic effect when a threshold or over- TABLE9 threshold dosage of colchicine is used, the members of the second can activate even an underthreshold SYNERGISTSOFCOLCHICINE dosage of colchicine. As an example of the first bulbocapnine+corydine +glaucine group, data on bulbocapnine are presented in +laurotetanine Table 7, and, as an example of the second group, +laurotetanine +methyl TABLE 7 etherapomorphine â€”morphothebaine SYNERGISTICEFFECTOFBULBO â€”corytuberine CAPNINEONCOLCHICINE â€”boldine â€”N-acetyllauro- jColchicine0.0050.00550.0060.0080.010.02Â»O/MLBulbocapnine102040HO888858PERCENTAGEOFDOSAGE â€”tetaninechelerythrine MITOSES1.52.21.82.81.11.7e. +-t- Â»=synergistic.â€” â€¢Â»notsynergistic.papaverinetetrahydro-papaverineberberinetetrahydro-berberinemorphinecodeinethebained-tubocurarinequininestrychnineveratrine

s24.423.846.6 TABLE 10 SYNERGISTICEFFECTSINTISSUECULTURES OFSTEROIDHORMONESONCOLCHICINE Colchicine(Mi/ml)0.010.010.010.010 those concerning phlorhizin in Table 8. Bulbocap- Hormone* mitoses5.02.421.02.613.02.63.414.221.

TABLE8 testosterone testosterone SYNERGISTICEFFECTOFPHLO testosterone propionate RHIZINONCOLCHICINE testosterone propionate PERCENTAGE estrone /Colchicine0040.020.0110.010.0080.00550.00450.0030.0110.0110.0110.0110.0110.00550.00450.0031G/MLPhlorhitii37Â«1889447188940S1.5949494DOSAGE estrone or MITOSES 0160.0160.0240.0240.0050.00750.0100.0050.00750.0100.0050.00750.010Percentageof 1.9 estrone S42.562.22.02.55.51.22.090S.S8.231.3 2.0 2.0 estrone 1.8 1.6 54.6 27.2 desoxycorticosterone 6.4 acetate 5.5 desoxycorticosterone 35 acetate 2.2 desoxycorticosterone 2.6 acetate 2.4 cortisone 80.0 cortisone 47.2 cortisone 16.9 12.3 * Hormones added in solid form to the medium of the 6.6 tissue culture. 13.7 5.3 underthreshold dosage. Testosterone, for instance, 4.6 reinforces the activity of the threshold dosage of nine acts as a Synergist only with a dosage of col 0.01 jug/ml, while estrone is without any effect at chicine which is high enough to be effective alone, this level of colchicine. However, an overthreshold but phlorhizin can produce an effect with a dosage dosage of 0.016 Mg/ml colchicine can be activated of colchicine too low to be effective by itself. These by estrone (Table 10). Adrenal cortical hormones, substances and the other synergists have no ef both desoxycorticosterone and cortisone, are fect alone as mitotic poisons. Besides bulbocap pronounced synergists of colchicine. Unlike des nine, the alkaloids thebaine, glaucine, and lauro oxycorticosterone, cortisone is able to make effec tetanine are also highly effective synergists tive an underthreshold dosage of colchicine (Table 10). Phosphocreatine can counteract the syner- (Table 9).

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1952 American Association for Cancer Research. LETTRÃ‰â€”CytochemicalStudies of Cell Behaviorâ€”A Review 855 gistic effect of desoxycorticosterone, but not that tractile proteins. The synergistic effect of vera- of cortisone. trine, also a muscle poison, is antagonized by the These results can be interpreted by assuming addition of phosphocreatine. that colchicine is active in some way in the system These results suggest investigations concerning of contractile elements during cell division. The the role of steroid hormones in the metabolism contraction of the cell spindle and the pinching in and production of ATP. It is thought by us that of the cell during cell division are thus analogous steroid hormones have a "carrier function" in the to muscular contraction. It is remarkable that this processes of phosphorylation. analogy has been discussed since the end of the last Contrary to Ludford (82), who interprets the century by morphologists (see Heidenhain [18]), effect of colchicine from the morphological point but it was not until 1947 that the chemical anal of view as an inhibition of spindle formation, our ogy was discussed by BrÃ¤chet (3). He proposed results suggest only a dysfunction of the spindle. A the hypothesis that adenosine triphosphate (ATP) contractile system, a unit of form and function, is the source of energy for spindle contraction as may change its shape when its function is dis well as for muscle contraction. According to turbed. Real suppression of spindle formation is Szent-Gyorgyi (90), actomyosin, isolated from only brought about by factors which are also able muscle and precipitated in the form of threads, to inhibit blood clottingâ€”e.g., heparin (19) or can be induced to contract by the addition of substances able to bind calcium. ATP. The metabolism of the cell or the muscle Colchicine and N-ethylcolchicamide have been can produce the ATP needed for contraction used for local treatment of skin cancer, papilloma, either from phosphocreatine or from the degrada condyloma, and breast cancer by Brodersen (4). tion of carbohydrates by glycolysis or respiration. Colchicine has been applied in erythroplasia by Colchicine, however, inhibits respiration or gly SchÃ¶nfeld(87). Hirsch (20) described cases of pre- colysis only in amounts a thousand-fold higher cancerous nevi and rodent ulcers which disap than the amount necessary for inhibiting mitosis. peared after treatment with a combination of bul- Therefore, its effect on mitosis cannot be ex bocapnine and colchicine. The great number of plained by its direct effect on metabolism. The combinations of synergists with colchicine gives a same statement can be made concerning phos- broad field for investigations in cases of skin phatases. ATPase of liver or muscle is inhibited cancer. only by extremely high amounts of colchicine (47, 52). We found, however, that ATP added to 3. CHEMISTRYOPCELLDIVISION the tissue culture medium counteracts the action We consider the study of mitotic poisons as a of colchicine for some hoursâ€”as long as ATP is tool to investigate reacting components during stable under these conditions (52). BÃ¡rÃ¡nyand mitosis, and we may assume that these compo Palis (1) showed that colchicine influences the nents are also important in uninfluenced cell divi decrease in viscosity of a mixture of actomyosin sion. During interphase the metabolism of cyto and ATP. It seems important that the effective plasm (i.e., respiration) predominates; in addition, dosages used by these authors are comparable to protein synthesis occurs in both nucleus and cyto those which inhibit mitosis. Thus, we may formu plasm. During early prophase, an increase in late the theory that colchicine inhibits a reaction nuclear material occurs. During prophase the between ATP and a contractile system of the chromosomes consisting mainly of desoxyribo- actomyosin type inside the cell during mitosis. nucleic acid become visible; chemically, this indi The activity of colchicine depends on the amount cates formation and polymerization of nucleotides. of ATP inside the cell: i.e., it is inversely propor The precipitation of nucleic acids in the form of tional to the amount of ATP. On the other hand, chromosomes seems to be a condition sine qua non its activity depends on the state of the contractile of the dissolution of the nuclear membrane. In the material. interphase nucleus, nucleotides and polynucleo- From this point of view, we are able to inter tides are attached to the nuclear membrane; in this pret the synergistic effect of phlorhizin by its stage, the membrane seems to have the character ability to poison phosphotransferases, thus de of a nucleoprotein stable against proteolytic creasing the level of ATP inside the cell. Bulbo- enzymes. If the nucleotides are removed, the capnine has not previously been known to have an remaining membrane is susceptible to proteolytic effect on the production of energy-rich phosphate activity. When this does not take place, the bonds. It is a well known muscle poison which nuclear membrane cannot be dissolved, and the produces catalepsia; therefore, its synergistic nucleus can only divide directly by amitosis. We effect may be considered an effect on the con consider the formation of the cell spindle to be a

process similar and related to that of blood of ATP. Thus, the change from respiration to clotting. Spindle contraction, leading to the sepa glycolysis brings about a decrease in the ATP level ration of the chromosomes, is, as mentioned of the cell. We interpret the induction of motion of above, a process similar to muscle contraction. The the cell surface as a change from permanent con process of pinching in of the cytoplasm is a result traction to local periodic contractions. This means of the contractibility of the cell surface (43). that the surface of the "resting cell" is not in a real state of rest, but in a state of permanent 4. CELLMETABOLISM,GROWTH,ANDCELLDIVISION contractionâ€”a tonus, needing more chemical en In 1926, Warburg (92) summarized his investi ergy than the periodic contractions characteristic gation on cell metabolism and growth in the state of mitosis. In consequence, the induction of cell ment: "No growth without glycolysis." He found motion by poisons of respiration should be com that in malignant cells aerobic glycolysis is pre pensated by the addition of ATP; this was con dominant over respiration, in embryonic cells it is firmed experimentally. In agreement with Lewis comparable to respiration, but in adult normal (80), we consider the cell surface as a contractile cells glycolysis is less than respiration. Although layer and suggest that this layer has the qualities not every tissue with predominance of glycolysis of actomyosin; the stage of contraction depends must be a growing one, there is some relation be on the ATP level and thus on cell metabolism. tween growth and glycolysis. Growth is a two-step It seems of interest that the same hypothesis has phenomenon : the first step is the preparation of a been discussed by Goldacre (16) with regard to cell for division, and the second step is the division the movement of amoeba. As shown above, the cell itself. Thus, the question arises whether glycolysis spindle needs ATP for its contraction. During is related to the first or to the second step. The mitosis, there are at least two contractile systems: findings of O'Connor (84) on embryonic cells of (a) that of the cell surface, relaxed and needing different ages show that there exists a direct pro only a small amount of ATP, and (6) that of the portionality between the number of cell divisions spindle and related contractile fibers needing and aerobic glycolysis. A possible explanation ATP. The addition of ATP to a tissue culture of would be that during division glycolysis predomi fibroblasts reinforces both systems. We would nates, respiration being inhibited. expect to find that the cell surface cannot relax The results of the following experiments are in after treatment with exogenous ATP, and, indeed, agreement with this finding (48, 53, 54). In a most of such cells in metaphases are not round but tissue culture of fibroblasts, there are on the spindle-shaped, as are resting cells (Fig. 5). The average 98 per cent of cells in interphase and 2 per separation of the chromosomes by contraction of cent in the stage of division. During mitosis, the the spindle fibers is favored, and cell division fol spindle-shaped cells become spherical. Cell divi lows. From stained preparations it seems as if sion is characterized by movement of the cell cell division in many of these cases takes place by surface, which pushes out pseudopodia. From the tearing of the cells (Fig. 6) ; by direct observation morphological point of view we must consider this of living fibroblasts with ATP added, however, we stage of the moving cell as functionally connected have not yet been able to observe this type of with mitosis and the movement of chromosomes. division. This type of cell division in an unin But in motion pictures of cells under the action of fluenced cell could only be produced by a metabo mitotic poisons, we have seen that plasma motility lism with an extremely high production of ATP. occurs, although the separation of the chromo Together with the induction of plasma motion somes is inhibited (33). Furthermore, we were able in resting cells by treatment with respiratory to show that plasma motion can be induced by all poisons, we observed the pinching off of plasma factors inhibiting respiration but not effecting bubbles which then moved freely within the glycolysis. As the enzymes of respiration are most medium. It is believed that these moving lumps ly localized in the mitochondria, dyes with a more consist of a contractile layer and an enzyme or less pronounced affinity for mitochondria prove system producing ATP sufficient for local contrac to be highly effectiveâ€”e.g., victoria blue, janus tions but not for permanent contraction. It is green, and others. Poisons of respiration such as our opinion that this is the simplest biological potassium cyanide or the removal of oxygen have system able to move (by means of carbohydrate the same effect (reversible in the latter). metabolism), and we are engaged in attempts to The link between metabolism and motion ap combine actomyosin with enzyme systems, with pears to be the effect of ATP on contractile pro the aim of "synthesizing" a primitive protoplasm- teins. Respiration produces at least 12 moles of like system with motion and metabolism. ATP per mole of hexose but glycolysis only 2 moles In general, cell shape and stability of cell shape

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1952 American Association for Cancer Research. LETTRÃ‰â€”CytochemicalStudies of Cell Behaviorâ€”A Review 857 depend on the ATP level of the cell and thus on its water. If this process was repeated 3-4 times, some metabolism. If metabolism produces a sufficient of the swollen cells began to lose cytoplasm. This amount of ATP, permanent contraction of the removal of cytoplasm may be easily completed by surface is possible, and the cell shape is stable. A the addition of a surface-active substance, such as smaller amount of ATP results in periodic surface digitonin. This method enables us to obtain iso contractions, and the form of the cell is not stable. lated nuclei in a morphologically pure state (see Such an unstable system not only may move but Fig. 8). These nuclei produce no tumors after is forced to be in motion as long as the same meta transplantation, in agreement with the behavior bolic conditions obtain: this is uncontrolled mo of cell nuclei prepared by homogenization. Al tion. On the other hand, metabolism with high though pure nuclear fractions may be obtained production of ATP is able to control cellular in this way, this method is not suitable for chem motion. ical investigations on nuclei, because a great loss As far as the cancer cell is concerned, we can of substances must occur during the preparation. conclude that the greater part of glycolysis is During the repeated process of swelling, the connected with the interphase; we consider this as tumor cells lose granules from their cytoplasmâ€” related to synthetic processes (see Zamecnik et al. large as well as small ones. Thus, we can obtain [94]). The mitosis of cancer cells is characterized cells with a nucleus and cytoplasm, but with a by a short prophase and a long metaphase, while gradual loss of structural cytoplasmic components that of normal cells conversely has a long pro- (Fig. 7). If the swelling and centrifuging of the phase and a short metaphase (Koller [22]). We cells took place only 2 or 3 times, the cells were interpret the short prophase as the result of an still able to produce an ascites tumor identical to acceleration of synthetic processes in the cancer that from untreated cells. However, cells treated cell, the long metaphase as a result of a deficiency 4 times (with their cytoplasm almost free of par- in ATP (37). ticulates) gave no tumors after injection into mice. Thus, we can conclude that reconstruction 5. BEHAVIOROFCOMPONENTSOFTUMOKCELLS of a complete cell is impossible from a cell with IN TRANSPLANTATION nucleus and cytoplasm, but depleted of mito Since 1949, we have carried out experiments to chondria and microsomes. Although we must determine whether components of tumor cells are realize that the nucleus of these cells may be able to induce tumors and whether reconstruction damaged by the repeated process of swelling, we of a tumor cell out of its components is possible (38-42) (similar to Stasney et al. [89]). The can consider these findings as a hint that mito chondria can arise only from mitochondria. simplest method to get a complete mixture of all In the preparations of cells swollen 2 or 3 times, components is homogenization. We used a blendor the phase contrast microscope shows granules still at a speed of 18,000 r.p.m. The mouse ascites inside the cells as well as between the cells. The tumor, the Walker carcinoma, and the Jensen positive tumor-takes of these preparations sug sarcoma of the rat lost their ability to produce gested experiments on the behavior of mixtures of tumors when they were homogenized sufficiently particulate-free cells and fractions with particu- for complete cell destruction. With the Walker lates. Isolated particulate fractions of this tumor carcinoma, these findings are in agreement with produced no tumors and neither did particulate- those of Tourtelotte and Storer (91). From these free cells, as mentioned above; but when we in results, we can conclude that the reconstruction jected into the same mouse particulate-free cells of an intact cell out of its components is im and particulate fractions or a mixture of them, we possible, at least for the types of mammalian observed the production of tumors. We may tumors investigated and the conditions employed. evaluate such a result as positive only if the cell With the Rous sarcoma, however, we were able to fractions used in the experiment are unable to pro produce tumors with the homogenate. The virus duce tumors when injected alone. Thus, in every is not destroyed mechanically. The simplest method to distinguish between a "virus"-induced experiment controls with the individual fractions are necessary. Out of seventeen experiments, six tumor and a tumor transplantable only in the gave a positive result: the components alone gave form of intact cells seems to be this test of cell-free no tumor, the mixture produced tumors. In five homogenates for their ability to produce tumors. experiments, one component alone produced tu We developed a method to isolate nuclei from mors; therefore, these experiments cannot be the cells of the mouse ascites tumor. The cells evaluated. In six experiments we obtained tumors were placed in distilled water, in which they swell; neither from the components alone nor from the they were centrifuged and again placed in distilled mixture. Regarding the fragility of the fractions

and the many possibilities of injuring them in their rhythmus und auf die Zellteilung in Fibrocytenkulturen. purification, negative results are of a high prob Ztschr. f. Zellforsch, und mikroskop. Anat., 29:283-322, 1939. ability. If we consider the positive results to be 7. COLLIER,W. A., and .1MIN,G. Ãœberdie Natur des Ehr- reliable, there exists the possibility of recombina lich'schen MÃ¤usecarcinoms.Ztschr. f. Krebsforsch., 40: tion of plasma granules with particulate-free cells. 298-304, 1934. Mixtures of particulate-free cells and complete 8. COOK,J. W., and ENGEL,L. L. Colchicine and Related Compounds. Part II. Synthesis of a Simple Analogue of homogenates also resulted in positive takes. The N-Acetylcolchinol Methyl Ether. J. Chem. Soc., London, supernatant fluid of homogenates of tumor cells pp. 198-200, 1940. (free from structured particles) was unable to pro 9. DEWAH,M. J. S. Structure of Colchicine. Nature, 155:141, duce tumors when combined with particulate-free 1945. 10. . Structure of Colchicine. Nature, 166:479, 1945. cells. Thus, we think that the particles of the cyto 11. DUSTIN,A. P. Contribution Ã l'Ã©tudedes poisons caryo- plasm are important for the effect and not other clasiques sur les tumeurs animales. II. Action de la col- substances such as proteins or compounds of chicine sur le sarcome greffÃ©,typeCrocker, de la souris. lower molecular weight. At a temperature of +4 Acad. Roy. Med. Belg., 14:487-502, 1934. to 10Â°C.,the cells of the mouse ascites tumor are 12. EIGSTI,O. J., and DUSTIN,P., JR. Colchicine Bibliography. Lloydia, 10:65-114, 1947. stable for 6-8 days. At this temperature tumor 13. . Colchicine Bibliography. III. Ibid., 12:185-207, cells dialyzed against distilled water showed no 1949. loss of transplantability. This demonstrates that 14. FLEISCHMANN,W.,and ULLTOT,G. E. Studies on Colchi substances of low molecular weight able to dialyze cine Derivatives. II. Cancer, 3:130-33, 1950. 15. GODEAUX,J.Action des toxiques de guerre et des vesicants under these conditions can be reacquired from the sur les fib de myosine. Bull. Soc. Roy. Se., LiÃ¨ge,106:216- body fluids. 27, 1944. The possibility of recombining tumor cell frac 16. GOLDACHE,R.J. The Folding and Unfolding of Protein tions can be regarded as an interesting fact in Molecules as a Basis of Osmotic Work. Internat. Rev. cancer research, if these data are reliable. There Cytol. 1:135, 1952. fore, these experiments are now being repeated ; we 17. GOLDBERG,B.; ORTEGA,L. G.; GOLDIN,A.: ULLTOT, G. E.; and SCHOENBACH,E.B. Studies on Colchicine are trying to find reproducible conditions for the Derivatives. I. Cancer, 3:124-29, 1950. preparation of the cell fractions. The conclusions 18. HEIDENHAIN,M.Plasma und Zelle. Jena, 1907. from these results may suggest new aspects con 19. HEILBHUNN,L. V., and WILSON,W. L. The Effect of HÃ©parineonCell Division. Proc. Soc. Exper. Biol. & Med., cerning the mechanism of the formation of mÃ©tas 70:179-81, 1949. tases and recurrences: perhaps such fragments of 20. HIRSCH,H. Ãœberdie Wirkungssteigerung von Colchicin tumor cells can combine with scattered cells which durch gleichzeitige Anwendung von Bulbocapnin bei der have lost their capacity for growth and thus Behandlung von Hautkrebsen. Dermatol. Wchnschr., initiate neoplastic growth. An analogy is given to 123:389-90, 1951. the induction of tumors by virusesâ€”but no more 21. KLAGES,A. Einfluss organischer Quecksilberverbindungen auf die Zellkernteilung im pflanzlichen und tierischen than an analogy. The development of grafted Organismus. Angew. Chem., 68:41, 1945. tumors can be accelerated by injections of homog 22. KOLLER, P. C. Experimental Modification of Nucleic enates of the same tumor type. In the mouse Acid Systems in the Cell. Symp. Soc. Exper. Biol., 1:270- ascites tumor, a wave of mitoses occurs several 90, 1947. 23. LEHMANN,F.E. Chemische Beeinflussung der Zellteilung. hours after the injection of a homogenate of this Experientia, 3:223-32, 1947. tumor. Among the fractions of the homogenate, 24. LETTRE,H. Die GewebezÃ¼chtungals Hilfsmittel chemi the mitochondrial fraction is the most effective, scher Krebsforschung. Angew. Chem., 53:363-68, 1940. while the nuclei and the supernate are less effec 25. . Einige Beobachtungen Ãœberdas Wachstum des MÃ¤use-Ascites-Tumorsund seine Beeinflussung. Ztschr. f. tive. physiol. Chem., 268:59-76, 1941. REFERENCES 26. . Nachtrag zur Arbeit: Einige Beobachtungen Ãœber das Wachstum des MÃ¤use-Ascites-Tumors und seine 1. BÃ•RÃ•NT,E.,and PALIS,A. Hemmung des ViskositÃ¤tsab Beeinflussung. Ibid., 271: 190-91, 1941. falles in ATP-Actomyosin-Misehungen durch Colchicin. 27. . Zur \Virkung von Trypaflavin auf den MÃ¤use- Naturwissenschaften, 38:547, 1951. Ascites-Tumor. Ibid., pp. 192-99. 2. BAUCH,R. Trypaflavin als Typus der Chromosomengifte. Naturwissenschaften, 34:346-47, 1947. 28. . Hemmstoffe des Wachstums, insbesondere Mito segifte. Angew. Chem., 55:265-66, 1942. 3. BRÃ„CHET,J. Embryologie chimique. Paris: Masson & Cie, 1947. 29. . Mitosegifte und ihre Beziehungen zu Naturstoffen. 4. BRODERSEN,H. Mitosegifte und ionisierende Strahlung. Naturwissenschaften, 30:34-40, 1942. Strahlentherapie, 73:196-256, 1943. 30. . Hemmung der Zellteilung durch estrogene Fak 5. BRUES,A. M., and COHEN,A. Effects of Colchicine and toren. Ztschr. f. physiol. Chem., 278: 201-5, 1943. Related Substances on Cell Division. Biochem. J., 30: 31. . Ergebnisse und Probleme der Mitosegiftforschung. 1363-38, 1936. Naturwissenschaften, 33:75-86, 1946. 6. BÃœCHER,O.Zur Kenntnis der Mitose. VI. Der Einfluss 32. . Mitosegifte und cancerogene Faktoren als Anti von Colchicin und Trypaflavin auf den Wachstums biotica. Ztschr. f. Krebsforsch., 66:5-35, 1948.

88. - -. Mitose und Mitosegifte. Hochschulfilm C 599. 59. . Vergleich von Colchicin, Isocolchicin und Homo GÃ¶ttingen, Bunsenstr. 10, Germany: Institut fÃ¼rFilm logen auf ihre zellteilungshemmende Wirkung. Ibid., 289: und Bild in Wissenschaft und Unterricht. 1951. 123-27, 1952. 84. â€” . Teilung menschlicher Tumorzellen. Hochschul 60. LETTRÃ‰,H.,and HARTWIG,E. Ãœberdie Bedeutung des film C 600. GÃ¶ttingen,Bunsenstr. 10, Germany: Institut Tropolonrings im Colchicin fUr die antimitotische Wir fÃ¼rFilmund Bild, 1951. kung. Ztschr. f. physiol. Chem. (in press). 86. . ÃœberMitosegifte. Ergeh. Physiol., biol. Chem. 61. LETTRÃ‰,H.,and KRAMER,W. Eine gegen Colchicin exper. Pharmakol. 46:379-452, 1950. resistente Abart des MÃ¤use-Ascites-Tumors.Naturwissen 86. . Einige Versuche mit dem MÃ¤use-Ascites-Tumor. schaften, 39:117, 1952. Ztschr. f. Krebsforsch., 67:1-13, 1950. 62. LETTRÃ‰,H.; KRAPP, R.; and OCHSENSCHLÃ„GER,M. 37. . Die Teilung normaler und bÃ¶sartiger Zellen als Wirkungen des Colchicins und N-Methylcolchicamids auf biochemisches Problem. Strahlen thÃ©rapie,83:1-14, 1950. die Mitose der Zellen des MÃ¤use-Ascites-Tumors.Ztschr. 38. . Ãœber das Verhalten von Bestandteilen der f. Krebsforsch., 67:142-50, 1950. Tumorzelle bei der Transplantation. Naturwissenschaften, 63. LETTRÃ‰,H.;LANDSCHÃœTZ,C.;and NOBEL,J. ÃœberSyner 37:335-36,1950. gisten von Mitosegiften. X. Mitt. Nebennierenrinden- 39. . ÃœberdasVerhalten von Bestandteilen der Tumor hormone und Colchicin. Klin. Wchnschr., 29:555, 1951. zelle bei der Transplantation. II. Mitt. Verhalten homo 64. LETTRÃ‰,H.,and LETTRÃ‰,R.Aufhebung der Wirkung von genisierter Tumoren. Ztschr. f. Krebsforsch., 67:121- Mitosegiften durch chemische Faktoren. Naturwissen 24, 1950. schaften, 33:283-84, 1946. 40. . III. Mitt. Zellkerne, Plasmagranula und granula 65. . Aufhebung der Mitosegiftwirkung metallorga freie Zellen. Ibid., pp. 345-52, 1951. nischer Verbindungen. Ibid., 34:127, 1947. 41. â€” . IV. Mitt. Dialysierte Tumorzellen. Naturwissen 66. LETTRÃ‰,H.;LETTRÃ‰,R.;and PFLANZ,C. VerstÃ¤rkungder schaften, 38:119-20, 1951. mitosehemmenden Wirkung des Colchicins durch kata- 42. . V. Mitt. Gemische von granulafreien Zellen und tonieerzeugende Stoffe. Naturwissenschaften, 37:378, Plasmagranula. Ztschr. f. Krebsforsch., 67:661-64, 1951. 1950. 43. . Physikalisch-chemische Betrachtungen Ã¼ber 67. . ÃœberSynergisten von Mitosegiften. II. Mitt. Wachstum und Zellteilung. Ztschr. f. Elektrochem. angew. Bulbocapnin, Colchicin und N-Methylcolchicamid. physikal. Chem., 56:531-35, 1951. Ztschr. f. physiol. Chem., 286:138-44, 1950. 44. . ÃœberSynergisten von Mitosegiften. V. Mitt. 68. . III. Mitt. Phlorrhizin, Colchicin und N-Methyl Versuche zur Aufhebung der synergistischen Wirkung colchicamid. Ibid., pp. 212-16. durch Phosphagen. Naturwissenschaften, 38:13, 1951. 69. . IV. Mitt. Naturwissenschaften, 37:563, 1950. 46. . ÃœberSynergisten von Mitosegiften. IX. Mitt. 70. . VI. Mitt. Aporphinderivate und Colchicin. Ztschr. Synergisten des Colchicins. Arzneimittelforschung, 1:3-5, f. physiol. Chem., 287:53-58, 1951. 1951. 71. . VII. Mitt. Weitere Alkaloide und Colchicin oder 46. . Synergists of Mitotic Poisons, Especially Steroid N-Methylcolchicamid. Ibid., pp. 150-57, 1951. Hormones and Colchicine, p. 294. New York: Xllth 72. . Vili. Mitt. Sexualhormone und Colchicin. Natur Internat. Congress Pure & Applied Chemistry. 1951. wissenschaften, 38:70-71, 1951. 47. . Zur Chemie und Biologie der Mitosegifte. Angew. 73. . XI. Mitt. Eine UnterscheidungsmÃ¶glichkeit von Chem., 63:421-30, 1961. Desoxycorticosteron und Cortison. Ibid., pp. 214. 48. â€¢ . Zellstoffwechsel und Zellteilung. Naturwissen 74. LETTRÃ‰,H.;LETTRÃ‰,R.;and RIEMENSCHNEIDEH,W. schaften, 38:490-96, 1951. Zur mitosehemmenden Wirkung des Adrenalins. Natur 49. LETTRÃ‰,H.,and ALBRECHT,M. Zur Wirkung von jS- wissenschaften, 38:282-83, 1951. Phenylaethylaminen auf in vitro gezÃ¼chteteZellen.Ztschr. 75. LETTRÃ‰,H.,and LUTZE,M. Beitrag zur Verteilung und f. physiol. Chem., 271:200-7, 1941. Wirkung des Colchicins im TierkÃ¶rper.Ztschr. f. physiol. 60. . PrÃ¼fungeiniger Alkaloide auf eine Mitosegift Chem., 281:58-64, 1944. wirkung. Ibid., 281:133-38, 1944. 76. LETTRÃ‰,H.;MATER,A.; and PFLANZ,C. Darstellung der 61. . Weitere Untersuchungen Ã¼bereine Mitosegift Zellspindel in Fibroblasten. Naturwissenschaften, 38:119, wirkung von Alkaloiden. Ibid., 287:58-65, 1951. 1951. 62. . Ãœberdie AbhÃ¤ngigkeitder Colchicinwirkung von 77. LETTRÃ‰,H.;MEX, A.; and RTTHBATJM,E.Vergleich homo der AdenosintriposphorsÃ¤ure. Naturwissenschaften, 38: loger 4'-Alkoxystilbylamine und optischer Antipoden auf 547^*8, 1951. ihre zellteilungshemmende Wirkung. Ztschr. f. physiol. 63. LETTRÃ‰,H.;ALBRECHT,M.; and LETTRÃ‰,R.Verhalten Chem., 289:119-23, 1952. von Fibroblasten unter aeroben und anaeroben Bedin 78. LETTRÃ‰,H.,and SCHLEICH,A. Zur Wirkung des Trypa- gungen. Naturwissenschaften, 38:504-5, 1951. flavins und verwandter Stoffe auf die Zellteilung des 64. . Zur AuslÃ¶sungvon Plasmabewegungen bei Ruhe MÃ¤use-Ascites-Tumors.Dissertation Heidelberg, 1949. zellen. Ibid., p. 505. 79. LETTRÃ‰,H.,and STRATMANN,M.Ãœberweitere einfache SB. LETTRE,H., and BERGDOLT,H.Die Wirkung fraktionierter Mitosegifte. Ztschr. f. physiol. Chem., 288:25-30, 1951. N-Methylcolchicamidgaben auf den MÃ¤use-Ascites-Tu 80. LEWIS, W. H. Cell Division with Special Reference to mor. Ztschr. f. Krebsforsch, (in press). Cells in Tissue Culture. Ann. New York Acad. Sc., 61: 56. LETTRÃ‰,H.,and DELITZSCH,J. Zur Mitosegiftwirkung 1287-94, 1951. einiger Stilbylaminderivate. Ztschr. f. physiol. Chem., 81. LOEWENTHAL,H., and JAHN, G. Ãœbertragungsversuche 281:139-42, 1944. mit carcinomatÃ¶serMÃ¤use-Ascites-FlÃ¼ssigkeitundihr Ver 67. . Weitere Untersuchungen zur Mitosegiftwirkung halten gegen physikalische und chemische Einwirkung. von Stilbylaminderivaten. Ibid., 289:220, 1952. Ztschr. f. Krebsforsch., 37:439-47, 1932. 68. LETTRÃ‰,H.,and FERNHOLZ,H. Beitrag zur Beziehung 82. LUDFORD,R.J. The Action of Toxic Substances upon the zwischen der Mitosegiftwirkung und der Konstitution von Division of Normal and Malignant Cells in Vitro and in Colchicinderivaten. Ztschr. f. physiol. Chem., 278:175- Vivo. Arch. f. exper. Zellforsch., 18:411-41, 1936. 200, 1943. 83. MclLWAiN, H. A. Nutritional Investigation of the Anti-

bacterial Action of Acriflavine. Biochem. J., 36:1811-18, duction of Neoplasms by Injection of Fractions of Mam 1941. malian Neoplasms. Cancer Research, 10:775-82, 1950. 84. O'CONNOR,R. J. The Metabolism of Cell Division. Brit. J. 90. SZENT-GYORGYÃ•,A.Chemistry of Muscular Contraction. Path., 31:390-96, 1950. New York, 1947. 85. RAPKINE,L. Sur les processus chimiques au cours de la 91. TOURTELOTTE,W. W., and STOREH,J. B. The Role of division cellulaire. Compt. rend. acad. Se., 191:871-74, Cellular Fractions in the Transplantation of the Walker 1930. Rat Carcinoma 256. Cancer Research, 10: 783-84, 1950. 86. SANTAVT,R.,and REICHSTEIN,T. Isolierung neuer Stoffe 92. WARBURG,O.ÃœberdenStoffwechsel der Tumoren. Berlin, aus dem Samen der Herbstzeitlose. Helvet. chim. Acta, 1926. 33:1606-27, 1950. 93. WINDAUS,A. Untersuchungen Ãœberdie Konstitution des 87. SCHÃ–NFELD,J.H. Colchicinbehandlung der Erythroplasie. Colchicins. Liebigs Ann. d. Chem., 439: 59-75, 1924. Dermatol. Wchnschr., 124:837-39, 1951. 94. ZAMECNIK,P.C.; LOFTFIELD,R. B.; STEPHENSON,M.L.; 88. SORKIN,M. Iso-Colchicin. Helvet. chim. Acta, 29:246-48, and STEELE, J. M. Studies on the Carbohydrate and 1946. Protein Metabolism of the Rat Hepatoma. Cancer Re 89. STASNET,J.; CANTAROW,A.;and PASCHKIS,K. E. Pro search, 11:592-602, 1951.

FIG. 1.â€”Untreated culture of mesenchymal fibroblasta. Living cells. X240. FIG. 2.â€”Chicken heart fibroblasts 24 hours after the addi tion of 0.1 ng/m\ N-propylcolchicamide. Living cells. X240. FIG. 3.â€”Untreated fibroblasts in metaphase. Ehrlich- Biondi stain. Phase contrast microscope. X540. FIG. 4.â€”Cellsof the mouse ascites tumor 24 hours after the injection of 4 pg. N-methylcolchicamide. Arrested metaphases with scattered chromosomes. Feulgen stain. X650. FIG. 5.â€”Fibroblasts 3 hours after the addition of 1 mg/ml ATP (sodium salt). Ehrlich-Biondi stain. Phase contrast microscope. X480. Metaphase in a triangular cell, telophase in an elongated cell. FIG. 6.â€”Sameconditions as Figure 5. Type of a cell division by "tearing." A metaphase in a spindle-shaped cell. FIG. 7.â€”Cellsof the mouse ascites tumor deprived of gran ules (prepared by swelling, repeated 4 times). Compare the cell size with Figure 4. Phase contrast microscope. X630. FIG. 8.â€”Isolated nuclei from swollen cells. X630.

Hans Lettré

Cancer Res 1952;12:847-860.

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