NUCLEAR ABNORMALITIES RESULTING FROM INHIBITION OF MITOSIS BY COLCHICINE AND OTHER SUBSTANCES ' AUSTIN M. BRUES AND ELIZABETH B. JACKSON (From the Medicul Lnborutorif~ of the Collis P. Iiz4ntingtolz M~moricil Hospitul of IIavvard University) Attention has been called, during the past few years, to the striking phenomenon of mitotic inhibition which occurs as a result of injecting certain substances into living animals and tissue cultures. The most striking effects of this sort occur, as originally described by Dustin (I), following treatment with sodium cacodylate and with the alkaloid colchicine. After the admin- FIG. 1. AREA IN PERIPHERY OF SARCOMA 180 TREATED WITH COLCHICINE Four mitoses can be seen. All sections are stained with iron-hematoxylin and eosin. X 1000. istration of these substances, large numbers of abnormal mitotic figures appear in all tissues, both normal and malignant, in which cell divisions normally occur in appreciable numbers. There is general agreement that these abnormal figures are characterized in all cases by the absence of the spindle, and Ludford (2) has attributed the suspension of division to the failure of that mechanism of which the spindle is the visible attribute. If we examine a slide of mouse sarcoma No. 180 from an animal which was injected with 0.025 mg. of colchicine ten hours previously, we see that about 12 per cent of the cells are in mitosis, in contrast to the normal figure of about 1 or 2 per cent (Fig. 1). When viewed superficially, many of these figures are not very different in appearance from normal figures, but upon careful observation it appears that there is no vestige of a spindle mechanism Read before the American Association for Cancer Research, Chicago, Ill., March 24, 1937. For discussion see p. 589. 504
INHIBITION OF MITOSIS BY COLCHICINE 505 TABLE I: Percentage of all Cclls in Sarcoma 180 in Varioz~s Pkascs of Mitosis (4000 cells were counted in each group) - -- 1 --- Percentage of All Cells 1 1 1 Colchicine (Periphery)......... 0.88 11.18 0.22 0.12 12.40 Colchicine (Center)............ 0.19 0.28 0.15 2.30 Co~~tro,Tutnor... 0.30 i: 0.14 0.14 0.0% in the great majority of these cells, that the incidence of late stages (anaphases and telophases) is relatively much reduced, and that the chromosomes, although usually clumped together in a position suggestive of the normal early metaphase, are frequently somewhat scattered and clumped into many groups of different sizes. Detailed examination of this tumor shows certain note- Surface of Tumor FIG. 2. MAP OF SECTION TIIROUGII A COLCIIICINE-TREATED SARCOMA 180 Figures show percentages of cells in mitosis in the marked areas. worthy features. In the first place, the incidence of arrested mitotic figures is much higher around the periphery of the tumor than it is near the center, remote from the blood supply. A diagram of this tumor shows the percentage of dividing cells in nine selected areas, in each of which about 1000 cells were counted. The frequency is seen to be relatively low at the extreme periphery, where there are some normal cells mingled with sarcoma cells (Fig. 2). It reaches a maximum just below this point, and then shades off to figures in the center of the tumor which are not unlike those found in normal tissues. This may be due either to the fact that mitosis is less prevalent in the central portion of the tumor, normally, or that the drug fails to penetrate the entire 5 or 6 mm. of tissue; actually both factors are probably active. The incidence of the different phases of mitosis was observed in three groups of 4000 cells each: in the areas of this tumor showing high and low frequencies of division, and in a control tumor in an untreated animal in the
5 06 AUSTIN M. BRUES AND ELIZABETH B. JACKSON same series of transplants (Table I). If the arrested figures are classed as metaphases, which they nlost closely resenlble, it appears that the actual incidence of this phase is significantly increased in the peripheral area, while the number of late phases, as an absolute percentage of all cells, remains nearly the same. In the center of this tumor the picture is practically the same as in the untreated tumor, which suggests that the low incidence is due largely to the fact that the drug does not act in the deeper part of the tumor. In the course of assaying certain derivatives of colchicine (3), the hypertrophying rat liver, after partial hepatectomy, was found to be a very suitable organ for studying mitotic arrest, because of the large size of cells and nuclei, the high frequency of mitosis, and the uniformity of distribution of both normal and abnormal figures throughout the organ. The remainder of this study has, therefore, been made on animals in the process of recovery from partial removal of the liver. The technic of operation and a statistical analysis of cell division in this preparation have been given in previous papers (4, 5). Since mitosis does not begin in any great proportion of the cells until a day after the operation, colchicine has been given by s~~bcutaneous injection from twenty-five to thirty hours after operation. The arrested mitosis in the hepatic cell differs from that in most other cells (including sarcoma cells) in that the chromosomes are widely scattered throughout the cell (Fig. 3). They appear to be somewhat shortened and thickened, in a cytoplasm which is more homogeneously staining than that of the surrounding cells, and the cell borders tend to be somewhat rounded. (The last two noted changes are characteristic of normal dividing cells, but in less degree than in the arrested cells.) When smears of these cells are made, a chromosome count of about 35 or 36 is found, although the majority
INHIBITION OF MITOSIS BY COLCHICINE 507 of arrested cells rupture in making a tissue smear (Fig. 4), while normal resting cells and untreated dividing cells show little tendency to rupture under these conditions. Livers with 20 per cent or more of arrested mitoses are grossly extremely friable and easily torn apart. These observations suggest that changes in the physical condition of the cytoplasm may be correlated with mitotic arrest. With doses from 0.01 to 0.2 mg. of colchicine per 100 grams body weight, large numbers of these abnormal figures are seen. With larger doses (above 0.2 mg. to as high as 10 mg.) mitosis is apparently prevented from beginning, as only a few abnormal figures are seen in numbers which correspond to the number of cells which might have been in mitosis at the moment the drug was given. As might be expected, on this basis, when the drug is given just before the onset of mitosis (at about twenty-four hours after operation) the figures fail to appear at all. These larger doses usually prove fatal after a few hours. With smaller doses (0.001 to 0.01 mg.) partially abnormal figures are seen, in which aberrant chromosomes or groups of chromosomes frequently appear at a distance from the main group (Fig. 5). It is worth remarking that in this tissue (and in most normal tissues) the late stages of mitosis are not seen at all in the first hours of treatment, contrary to what occurs in tumors. Examination of sections from over 100 animals receiving doses of 0.01 to 0.2 n~g. failed to show a single normal anaphase or telophase in the first twenty-four hours. During the first few hours after administration of such doses, the percentage of hepatic cells in mitotic arrest mounts up as more cells go into division, until at twelve hours the figure is between 25 and 30 per cent. No further increase in percentage occurs after this unless a successive dose is given; and it appears that, in the case of a single dose, recovery sets in at about this time.
5 08 AUSTIN M. BRUES AND ELIZABETH B. JACKSON The process of recovery appears to pass through a bizarre series of nuclear figures. After about twelve hours, two changes in the chromosomes can be observed. The individual chromosomes become obviously shorter and thicker than they were at first, and tend.to clump together into larger or smaller groups (Fig. 6). At about eighteen hours, many cells are found which are FIG. 6. SWELLING AND AGGLUTINATION OF CIIROMOSOMES, EIGHTEEN HOURS. X 1000 loaded with miniature nuclei, and all stages of chromosome swelling from the earliest increase in size up to the full-sized micronucleus can be observed (Fig. 7). We interpret this as indicating that the micronuclei are formed by the swelling of individual chromosomes. Where there is an agglutinated group of chromosomes, they appear in similar fashion to form larger micronuclei.
INHIBITION OF MITOSIS BY COLCHICINE 509 At a later period, eighteen to twenty-four hours, these types of figures are still seen, but in addition to these binucleate and trinucleate cells can be seen, as well as cells with single ameboid-shaped nuclei (Fig. 8), the conformation of which suggests that they may represent early fusion of micronuclei to form a single resting nucleus. These figures, which have departed in this way from the stage of complete arrest, cannot be brought back to this stage by an additional dose, as sections taken two to four hours after such an additional dose show a very similar recovery picture to that seen twenty-four hours after the first dose and before the second is given. By forty-eight hours after injection, various stages of arrest and recovery are still seen but in greatly diminished numbers, and after this time normal metaphases, anaphases, and telophases may be observed concurrently with the recovering abnormal figures. At later periods, there remain rare recovery forms, and there is a high incidence of binucleate cells through the third day. Examination of other normal tissues is less satisfactory, possibly because the process of transformation of small, closely agglutinated chromosomes into a single nucleus would be difficult to observe directly in fixed material. Telophases seem to apbear earlier in the intestinal crypts (twenty-four hours) than they do in regenerating liver, but whether this represents a different method of recovery from mitotic arrest or earlier removal of the drug locally from the tissue, it is impossible to say. Wholly similar pictures are seen following treatment with various derivatives of colchicine. Sodium cacodylate gives the same picture in complete mitotic arrest, but recovery appears to be slower, commencing at about twenty-four hours. DISCUSSION In attempting to evaluate the meaning of the various abnormal nuclear pictures seen, a chart has been made (Fig. 9) showing the incidence of each
5 10 AUSTIN M. BRUES AND ELIZABETH B. JACKSON stage at intervals following the administration of colchicine (0.1 mg. per cent). It appears from this that stages 1, 3, and 5 make up the preponderant number of abnormal figures at twelve, twenty-four, and forty-eight hours, respectively, and hence it is suggested that a single arrested mitosis may pass successively through these stages while in the course of recovery. Stages 2 and 4 are seen with somewhat less frequency, and presumably represent either phases which are passed through rapidly, or phases through which all of the cells do not The height of each black column represents the percentage of hepatic cells showing a given mitotic figure at each time period. For the sake of comparison, the first column in the twelvchour period represents 21 per cent, the second 10 per cent, and the third 2 per cent. The various figures have been classed in numbered groups as follows: 1. Chromosomes completely scattered. 2. Chromosomes somewhat agglutinated. 3. Micronuclei. 4. Amehoid nucleus. 5. Binuclear or trinuclear cell. 6. Partial spindle formation. 7. Complete spindle formation (normal mitosis). pass. Stage 6 (that of partial spindle formation), which appears only at the time when stage I has practically disappeared, is in no way different from the type of figure seen when a minimal amount of drug is administered, and so probably appears at the time when the amount of effective drug in the tissue is reduced to a corresponding level. Stage 1, that of colnplete scattering of chrornosoines, appears at once or within an hour after parenteral administration of colchicine, while stage 3, that of micronuclei, begins to appear about twelve hours later, and stages 4 and 5 after eighteen hours. Stage 1 disappears after forty-eight hours, and by
INHIBITION OF MITOSIS BY COLCHICINE 511 seventy-two hours all of the first four stages have disappeared. If, therefore, we are correct in supposing that these abnormal figures are stages in the process of recovery from initial arrest, it would seem that these stages are passed, in the case of a given cell, in the course of approxiniately twenty-four hours. Our observations on the nuclear pictures seen in sarconla 180 are a little at variance with those in liver. It seems that in this tumor, even in the periphery, where arrested Inetaphases are present in highest numbers, a fair proportion of anaphases and telophases can be found. We are unable to say whether this represents the arrest of a few cells in these late stages of mitosis at the time of drug administration, or whether it represents a form of recovery in which the normal course of mitosis is resumed. Investigations have been begun in this laboratory to test the effect of repeated injections of colchicine on the rate of cell increase over long periods of time. 1. Single injections of colchicine have been given to rats following partial hepatectoniy. This treatment causes an arrest of normal cell division which occurs at about the beginning of metaphase. Large numbers of abnormal figures therefore occur in the rapidly dividing hepatic cells of these animals. They appear at first in clear, rounded, readily ruptured cells, in which the spindle cannot be seen, and in which the chron~osomes are widely scattered. 2. Between 12 and 48 hours after the drug is given, large nunlbers of cells with bizarre nuclear figures appear, notably cells with numerous miniature nuclei, and cells with single large ameboid-shaped nuclei. It is suggested that these figures represent a specialized mode of recovery from arrested mitosis, in which individual chromosomes or small groups of chromosomes swell to form miniature nuclei, which in turn fuse to form one or more resting nuclei. 3. In the case of sarcoma 180, complete arrest of mitosis at early metaphase is not seen, in contrast to what occurs in liver. In all preparations studied, a few cells can be seen in later stages of division. In some grafted tumors the toxic effect of colchicine on mitosis is evident only around the margin of the tumor. BIBLIOGRAPHY 1. DUSTIN. A. P.: Action de la colchicine sur le sarcome greffc, type Crocker, de la souris, Bull. Acad. roy. de n1i.d. de Belgique 14: 487, 1934. 2. LUDFORD, R. J.: Action of toxic substances upon the division of normal and malignant cells in vitro and in vivo, Arch. exper. Zellforsch. 18: 411, 1936. 3. BRUES. A. M., AND COIIEN, A,: Effects of colchicine and related substances on cell division. Biochem. J. 30: 1363, 1936. 4. BRUES, A. M., AND MARBLE, B. B.: An analysis of mitosis in liver restoration, J. Exper. Med. 65: 15, 1937. 5. BRUES, A. M., DRURY, D. R., AND BRUES, M. C.: A quantitative study of cell growth in regenerating liver, Arch. Path. 22 : 658, 1936.