NUCLEAR ABNORMALITIES RESULTING FROM INHIBITION O F
MITOSIS BY COLCHICINE AND OTHER SUBSTANCES '
AUSTIN M. BRUES
AND
ELIZABETH B. JACKSON
( F r o m 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
505
INHIBITION OF MITOSIS BY COLCHICINE
TABLE
I : Percentage of all Cclls i n Sarcoma 180 in Varioz~sPkascs of Mitosis
(4000 cells were counted in each group)
--
---
1
Colchicine (Periphery). . . . . . . . .
Colchicine (Center). . . . . . . . . . . .
C o ~ ~ t r o , T u t n.o.r. . . . . . . . . . . . .
1
Percentage of All Cells
0.88
0.19
0.30
11.18
i:
0.22
0.28
0.14
1
0.12
0.15
0.14
1
12.40
2.30
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 O F SECTIONTIIROUGII A COLCIIICINE-TREATED
SARCOMA
180
Figures show percentages of cells in mitosis in the marked areas.
worthy features. I n 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 a t the extreme periphery,
where there are some normal cells mingled with sarcoma cells (Fig. 2 ) . I t
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
506
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 a t 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). I t 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).
I t 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 1 0 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 a t 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 a t 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. I t 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~osomesare 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. I t 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., A N D 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., A N D BRUES,M. C.: A quantitative study of cell growth in
regenerating liver, Arch. Path. 2 2 : 658, 1936.