A Study of the Mechanisms of Polyploid Nuclei Formation Induced in Tadpoles by Carcinogen-Protein Conjugates E. MARIEHEARNECREECH (The lnstitutefor Cancer Research and the Lanlèenau Hospital Research Institute, Philaddphia, Pa.) In earlier work, this author studied the action mechanisms involved in polyploid formation led of the choleic acids of some polycyclic aromatic to the consideration of problems of C-mitosis, en hydrocarbons on tissue cultures of mouse fibro doreduplication, and endomitosis. This paper is blasts (35—37). These compounds, which are concerned chiefly with those experiments which, water-soluble and possess known carcinogenic or through application of the most effective concen noncarcinogenic properties, produced definite dif trations of conjugate at the most appropriate ferences in chromosome behavior. The similarity stages of development, resulted in the greatest of their effects on chromosomes to those caused by production of mosaic animals with polyploid and the protein conjugates of carcinogens and noncar aneuploid nuclei. A definition of the terms as they cinogens used in the present study is striking. The will be used is given below. protein conjugates were used because of their C-Mrixyric Tnns solubility in water and because of their chemical All chromosome changes due to total or partial inhibition of structure, in that positions on the molecule of the the spindle are included in this category. carcinogens considered to be of importance to car a) Complefe C-niitosi,.—-This is Characterized by strong cinogenic activity are still free to react. The less chromosome contraction, beginning in prophase. Following the soluble amino acid conjugates of certain hydro disappearance of the nuclear membrane, the chromosomes are carbons are carcinogenic to mice, but the protein scattered irregularly throughout the cell. No metaphase plate is formed, and no anaphase movement occurs. Full C-mitoeis conjugates of the carcinogens have not produced may result in the formation of one or more telopizase nuclei, tumors in mice or rabbits. This is not unexpected, depending on the position of the chromosomesat the onset of because the antigenic properties of the latter telophase. No cleavage of the cytoplasm follows a full C-mite might counteract any carcinogenic potentiality of sis, and the cell consequently doubles its chromosome number. If two or more nuclei form, they may fuse later. the same molecule (16). Frog embryos do not form b) Partial C-mi2osia.—This involves some degree of spindle antibodies upon stimulation with foreign tissue action. The spindle is often either monopolar, resulting in nu proteins (34) and thus might be expected to re dci with one or more groups of chromosomes the halves of spond to any carcinogenic activity of the hydro which do not undergo anaphase movement, or multipolar, in carbon part of the conjugate. The pronounced whichcaseanaphase movementoccursto more than two poles. The resultant telophase nuclei may contain less than the solubility of the conjugates in water makes them normal chromosome number. particularly advantageous for the present work. Concentrations of 10—20mg. of conjugate (1.8—3.6 ENDOMITOTIC TYPES mg. of the 9,10-dimethyl-1,2-benzanthracene com These include chromosome number changes due to one or ponent) per ml. of water are readily obtained (60). more reproductions between two normal mitoses or in the absence of mitosis. This doubling occurs within the nuclear Tumors have been produced in amphibians by the membrane independently of spindle action. Two main types use of 20-methylcholanthrene choleic acid (13) and are distinguishable: with 20-methylcholanthrene (12). Holtfreter (38) a) Endoniitosü.—(In the strict sense first described by reports that carcinogens in amphibians induce the Geitler, 97, 28.) This process is associated with the visible formation of normal structures in embryonic mitotic contraction of the chromosomes (endoprophase and endometaphase) followed by the falling apart of the double stages but initiate cancerous growth in larval number of chromosomal elements (endoanaphase and endo stages. telophase). The separation is complete, since the chromosomes The chromosome studies in this paper were of the following prophase do not exhibit paired arrangement. made in conjunction with studies on the arrest of b) Endoreduplication.—(In the sense used by Levan and Hausehka, 49.) This type is not accompanied by visible endo development in frog embryos made by Green (30) mitotic stages. Two, instead of one, reproductions of the and with quantitative studies of polyploidy in the chromosome thread take place during interphase. Either the tail epithelium (31, 32). The study of the mitotic centromeres do not divide until later, in which case the follow ing prophase will show quadripartite diplochromosomes, or the Received for publication July 26, 1957. centromeres divide before the following prophase, still leaving 78 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1958 American Association for Cancer Research. CREEcH—Polyploid Nuclei Formation in Tadpoles 79 the prophase chromosomesin pairs. If more than one extra lin-bichromate-aceticacid mixture for 1@hours and stained reproduction occurs, correspondingly higher multiples of the with either Galligher's Hematoxylin and Fast Green or by the prophase chromosomes will be formed. Feulgen procedure. The first tips were taken simultaneously with the transfer of the animals to spring water at Shumway's [email protected] OF CLEAVAGE stage 25 (67). The regenerated tips were taken at weekly inter This may give rise to chromosomedoublings that are not vals. This material was found to be favorable for the study of caused by spindle disturbances and may be followed by nuclear the action of compounds on cell division, because a direct sur fusion or by the formation of a common spindle at the next vey of the entire cell population is possible, a high rate of mito mitosis (15, 73). Fusion of daughter cellsbefore or after cyto sis exists, the cells are not cut or squashed, and conditions iden kinesis has been reported by Hsu and Moorhead (40) and tical to those existing in the whole animal are expressed in the leads to the same result. tip. For further description of experimental conditions, see Green (80) and Green and Herdeck (82). The methods of treat MATERIALS AND METHODS ment are summarized in Table 1. The embryos described in these experiments were treated with various concentrations of the following conjugates in RESULTS aqueous solution: CowraoLs 9,10-dimethyl-1,9,-benzanthryl-8-carbamido bovine serum Untreated controls from the same lot of eggs @ albumin (9,10-dime-S-BovA-D42 and -1)27) 9,10-dimethyl-I,2-benzanthryl-S-carbamido horse serum were kept under the same laboratory conditions, I albumin (9,I0-dime-S-HSA-D27 and -42) and tail tips were taken at identical times and ex TABLE 1 SUMMARY OF EXPERIMENTS EXPERIMENT 1 Seriea A Series B Series C EXPERIMENT49,10-dime SEXPLRWzNT SEXPERnIENT Substance: 9,10-dime-3- Same Same BovA-D42 3-HSA-42 5-HSA-D27 S-HSA-D27 Embryos cx- Dark Dark Dark Dark Light1,Light9,10-dime Dark Light9,10-dimeDark posed in: Days exposed: 7 7 7 7 3 and 6 hours) 7 Embryos tram- Light Dark Light Dark Light(4 Dark Light. Dark Light ferred to: Conc. of hydro carbon (pg/mi): 4 4 9.5 9.5 9 . 16 2 No. treated: 5 5 5 5 15 4 10 No. controls: 5 5 5 5 15 4 10 Stage of 18 18 18 18 18 tips regen. 18 embryo:* at at.25 Weekly 5—7 1—S 1—S 1 (diedin 1—3 (diedin 3+ 5 10 cuttings: 2d week) 4 days) * Shumway stage 18: 96 hours at 18° C., 4 mm. long. Shumway stage 25: 284 hours at 18°C., 11 mm. long. 1,2-benzanthryl-10-carbamidohorse serum albumin (10- amined on successive weeks for each series in each HSA-16) experiment. Occasionally, chromosome irregulari S,4-benzpyrenyl-5-carbamidobovine serum albumin (BP BovA-I9) ties were found in the controls, but they were low 1,2,5,6-dibenzanthryl-9-carbamido horse serum albumin in frequency compared with the treated embryos. (DBA-HSA-0) They consisted of lagging and aberrant chromo @-anthryl carbamido horse serum albumin (fi-HSA-9) somes, chromosome bridges and fragments, and The synthesis and properties of these conjugateshave been clumped and abortive mitoses. One or two chromo described by Creech ci a!. (16—19)and Peck, Miller, and Creech somes were found sometimes in a separate vesicle (60). In the coding system, the final number represents the number of hydrocarbon groups per molecule of protein, and the in both control and treated animals. Triploid dlvi addition of the letter D means that the preparation was made sions and a rare haploid division were found in the in the absenceofactinic light to prevent photo-oxidationofthe controls, but these did not exceed 8 or 4 per cent. sensitive 9,10-dimethyl-l,2-benzanthryl component. Concen A normal diploid prophase is shown in Figure 6; trations are expressed in terms of the amount of hydrocarbon a normal polar metaphase in Figure 9 and a nor component/mi of solution. Eggs of the leopard frog, Bans pipien., obtained by inject mal bipolar telophase in Figure 18. ing female frogs with pituitary glands, were inseminated with sperm from macerated testes of the same speciesand kept in a Ex@m@i@r 1, SEm@s A room at 18°—19°C. This series was treated with 4 @g/mlof 9,10- Althoughseveralhundred tail tips from untreated embryos dime-3-BovA-D42 in the absence of actinic light and from embryos treated with different concentrations of other compounds were examined in preliminary studies, the and after 7 days was transferred to laboratory following four experiments were used for the detailed cytologi illumination (Table 1).