[CANCER RESEARCH 32, 746-755,
April 1972]
Mitosis in Human Leukemic Leukocytes during Colcemid
Inhibition and Recovery1
Manley McGill and B. R. Brinkley2
Department of Biology, The University of Texas at Houston, M. D. Anderson Hospital and Tumor Institute and Graduate School of Biomédical
Sciences, Houston, Texas 77025
SUMMARY
Normal stimulated lymphocytes and peripheral leukocytes
from five chronic and two acute myelogenous leukemia
patients were treated with Colcemid, 0.02 jug/ml, for 2 hr,
arresting dividing cells in a C-metaphase configuration. Upon
removal of Colcemid, cells progressed through metaphase,
anaphase, and telophase during time intervals from 0 to 120
min. Normal and leukemic leukocytes in various stages of
recovery were flat-embedded
for analysis by electron
microscopy. As in other mammalian cells, Colcemid inhibits
the migration of centriole pairs to opposite poles and results in
the formation of a unipolar spindle with chromosomes
displaced radially about the centrioles. Upon removal of
Colcemid, the centriole pairs moved apart and a normal
bipolar spindle was formed. The recovery of normal stimulated
lymphocytes proceeded rapidly up to 120 min. There was a
gradual decrease in the percentages of C-metaphase cells with a
subsequent increase in appearance of telophase cells.
The recovery of chronic myelogenous leukemia cells from
Colcemid block proceeds at a rate much slower than normal
lymphocytes. The percentages of telophase cells at 120 min
was low and most of the cells at this point remained in
C-metaphase.
Mitotic abnormalities
such as multipolar
spindles, anaphase bridges, and lagging chromosomes were
evident in some cells. Cells from three cultures of one patient
with acute myelogenous leukemia failed to recover from
Colcemid inhibition. Leukemic cells appeared to display a
greater sensitivity to Colcemid. Specific defects were noted in
the mitotic apparatus of leukemic leukocytes, including
abnormal centriole migration and atypical fine structure.
INTRODUCTION
Cytological analysis of leukemic leukocytes has been
reported by many investigators (3, 9, 10, 15). The results of
these studies have shown very little differences in normal and
leukemic leukocytes. In a review, Hayhoe (14) concluded that
the results of all the early studies of leukemic leukocytes failed
to show any consistent differences in the behavior of leukemic
'This work was supported in part by NIH Grants GM 15887, CA
05047, and CA 06939-05.
7To whom reprint requests should be addressed at Department of
Human Biological Chemistry and Genetics, the University of Texas
Medical Branch, Galveston, Texas 77550.
3The abbreviations used are: CML, chronic myelogenous leukemia;
HPMA, hydroxypropyl
methacrylate; AML, acute myelogenous
leukemia; MVB, multivesicular bodies.
Received October 15, 1971 ; accepted December 28, 1971.
746
and normal leukocytes in culture regarding their capacity for
differentiation
and early mitotic activity. More recently,
Rondanelli et al. (25), using phase contrast cinematographic
techniques, have shown that acute leukemic cells require a
much longer time to proceed through mitosis than normal
granulocytopoietic cells. Similar observations have been made
by other investigators (13, 20, 22), who used time-lapse
cinematography to study the duration of the mitotic cycle in
other cancer cells. Richart et al. (22), using cultured cells from
normal human cervical epithelium, dysplasia, and carcinoma in
situ, reported that the conduct of mitosis was strikingly
aberrant in the neoplastic cells and these cells spent
significantly more time in mitosis. They concluded that the
mitotic abnormalities seen in neoplastic cells represent a basic
alteration in the mitotic events occurring in the earliest stages
of the neoplastic transformation. This study confirms the early
work of Moorhead and Hsu (20), who used HeLa cells and
time-lapse techniques. These investigators reported that a
positive correlation exists between the increase in mitotic
duration and frequency of aberrancy in their HeLa cell
cultures (20). The increased incidence of mitotic error in
cancer cells may be due, in part, to abnormalities in the
mitotic apparatus. Therefore, the present study was designed
to: (a) compare the ultrastructure of the mitotic process in
normal and neoplastic leukocytes in vitro, and (¿>)
compare the
recovery rates of dividing normal and leukemic leukocytes
following inhibition with Colcemid, a well-known mitotic
inhibitor.
MATERIALS
AND METHODS
Leukocyte Cultures. Leukocytes were obtained from
peripheral blood cultures of healthy individuals and patients
with acute and CML.3 Blood specimens were collected by
venipuncture, with heparin as an anticoagulant. After the red
cells had settled for 2 hr, leukocytes were removed with the
plasma. A volume of plasma containing 10 to 40 X IO6
leukocytes/ml was placed into 10 ml of culture medium for a
final cell concentration of 1 to 4 X IO6 cells/ml of medium.
The culture medium used was FIO (Colorado Serum
Company, Denver, Colo.), containing 20% fetal calf serum
(Hyland Laboratories, Inc., Los Angeles, Calif.), plus 100 units
of penicillin per ml of medium. All cultures from normal
individuals received 0.2 ml of phytohemagglutinin M (Difco
Laboratories, Inc., Detroit, Mich.) per ml of medium, and all
cultures were incubated at 37°for 77 hr. Leukemic cultures
were incubated at 37°for 24 or 48 hr before treatment with
Colcemid. Colcemid was used to collect dividing cells in a
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Mitosis in Human Leukemic Leukocytes
C-metaphase configuration (4, 12), each culture receiving
Colcemid at a concentration of 0.02 fig/mi of culture for 2 hr.
Colcemid Experiments. Two hr after addition of Colcemid,
cultures were washed 3 times with phosphate buffer and
reincubated in fresh media. At time intervals from 0 to 120
min, samples of resuspended leukocytes were pelleted and
stained with acetic acid-orcein for light microscopy. The
number of C-metaphase, metaphase, anaphase, and telophase
cells per 100 mitotic cells was counted for each time period.
The mitotic index of each culture was calculated with the zero
minute specimen. In time periods where the mitotic index was
very low, 50 cells were scored. This procedure of reversal from
the effects of Colcemid is a modification of the procedure first
described by Stubblefield and Klevecz (28), using Chinese
hamster cells.
Light and Electron Microscopy. Cultured leukocytes for
light microscopic examination were fixed in 50% glacial acetic
acid and stained with 1% acetic acid:orcein stain. Cells in
acetic acid:orcein were dropped onto glass slides and slightly
pressed underneath the coverslip so that mitotic cells could be
viewed distinctly. Leukocytes for electron microscopy were
prepared by a modification of a procedure by Brinkley et al.
(5). Cells were fixed in a 3% glutaraldehyde buffered with
Millonig's phosphate buffer (19) at pH 7.4, postfixed in a 2%
solution of uranyl acetate, and rapidly dehydrated in a graded
series of ethanol. Two 5-min changes of 90% HPMA, one
3-min change of pure HPMA, and two changes of Luft's Epon
812: HPMA (1:1) are used before the cells are resuspended in
Luft's Epon mixture (18). Between each change of alcohol and
embedding media, the leukocytes were pelleted in a centrifuge
for 5 min at 1600 rpm. The pure Epon suspension of cells was
pipetted as evenly as possible on the bottom of a small plastic
dish, and more Epon was added to fill the dish to a depth of
about 2 to 4 mm. the Epon was polymerized at 60°for 48 hr.
The plastic dish was easily broken away from the polymerized
Epon and appropriate cells were located with a phase
microscope. The desired cells were bored out of the Epon disk,
mounted on a blank Epon capsule, and trimmed for sectioning
on an LKB Ultratome III. Serial sections were picked up on
collodian-coated, slotted grids (LKB, 2 x 1 mm), stained with
alcoholic uranyl acetate for 5 min, and poststained with lead
citrate (11) for 3 min.
RESULTS
Normal Lymphocytes. Upon removal of Colcemid from the
cultures, cells arrested in C-mitosis recovered to a normal
metaphase configuration and proceeded through anaphase and
telophase. This recovery process can be clearly shown by light
microscopy. Fig. 1 is a light micrograph showing the
C-metaphase configuration. Many of the chromosomes of this
cell seem to be held toward the center of the cell by their
centromere regions, and the chromatid arms appear to angle
back toward the outside of the cell away from the inside
kinetochores. Fig. 2 is an electron micrograph of another cell
in C-metaphase. In Fig. 3, a cell in an early stage of recovery
from Colcemid is seen in normal metaphase. Figs. 4 and 5 are
typical stages of anaphase and telophase seen in recovering
cultures. Examination
of cultures prior to removal of
Colcemid showed only cells in C-mitosis.
The percentages of C-metaphase, metaphase, anaphase, and
telophase configurations during recovery time periods was
made in normal, stimulated lymphocytes. All dividing cells
were arrested in C-metaphase and the Colcemid was removed
after 2 hr. In the 1st recovery experiments at 30, 60, 75, 90,
and 120 min, the number of C-metaphase, metaphase,
anaphase, and telophase cells was counted per 100 dividing
cells. Following removal of Colcemid, the percentage of the
dividing cells observed as metaphase cells reached a maximum
of 12% then declined. There was a gradual increase in anaphase
cells from 0 to 24% and a marked decrease in C-metaphase
cells from 100 to 36%. More significantly, there was an
increase in the number of telophase cells from 0 to 32%. These
results are reported as an average of 3 normal cultures
performed simultaneously. The 3 cultures were maintained
separately during 72 hr of incubation and then pooled
together for recovery. The mitotic index of the cultures was
4%. The data from a repeat experiment
of normal
lymphocytes showed a gradual decrease in the percentage of
C-metaphase cells with an increase in telophase cells to 31%.
The mitotic index of this culture was 6%. Table 1 is an average
of the percentage of 2 normal lymphocyte
recovery
experiments. At 60 and 90 min, the average plots include data
from one other reversal experiment where the mitotic
configurations were scored for those times only. The only
mitotic irregularity
seen during the light microscope
examination of normal cells was an anaphase bridge (Fig. 6)
seen in 1 cell.
CML. Six recovery experiments on multiple cultures of 4
CML patients have been completed. The percentages of
different mitotic figures at time intervals during recovery were
determined in the same manner as the normal recovery
experiments. Table 2 presents the percentages of C-metaphase,
metaphase, anaphase, and telophase cells scored after removal
of Colcemid from a CML leukocyte culture. C-metaphase cells
declined to 67% and the percentage of telophase cells at 105
min reached only 20%. The patient from whom this culture
was obtained was karyotyped as 46 chromosomes, including X
and Y and positive for the Philadelphia chromosome. Prior to
culturing, the patient had received daily drug treatments of
busulfan and had been diagnosed as a leukemia patient for 7
months. Table 3 presents percentages of mitotic configurations
seen during recovery of another CML patient. This patient had
no history of drug treatment for leukemia prior to cell culture
and had been diagnosed for 1 week. Telophase cells reached a
peak of only 11% during the 120-min recovery period.
Recovery experiments just completed were performed on
24- and 48-hr leukocyte cultures of another CML patient
without a history of leukemia or treatment for leukemia. In
the 24-hr culture, 120 min after recovery began, C-metaphase
cells declined to 62% and the percentage of telophase cells rose
to 18%. In the 48-hr culture, C-metaphase cells dropped to
36% at 120 min and telophase cells rose to 10% at 60 min and
dropped to 6% at 120 min. Table 4 is an average of the
percentages of mitotic configuration seen during time periods
of recovery from Colcemid block of 4 different CML cultures.
Each culture was performed in duplicate on recently diagnosed
CML patients with no history of drug treatment for leukemia
prior to culture.
Mitotic irregularities seen by light microscopy in recovering
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747
Manley McGill and B. R. Brinkley
Table 1
Recovery rate of normal, stimulated lymphocytes
Data reported are an average of the percentages of 12 normal
lymphocyte recovery experiments.
able to continue the division process leaving only the blocked
C-metaphase cells. In the leukemia cultures, Colcemid added
during mitosis may delay or inhibit anaphase and cytokinesis.
Ultrastructural
Studies. The typical arrangement
of
chromosomes in Colcemid-arrested cells has been shown in
Time interval
(%)C-metaphase100 configurations
Figs. 1 and 2. At a concentration of 0.02 Mg/ml, Colcemid
after Colcemid
removal(min)0
phase0 inhibits the formation of a metaphase spindle; thus, the
centriole pairs fail to separate and a unipolar spindle is formed.
Kinetochore microtubules from 1 sister kinetochore of each
1
3060°
9377
610
0
chromosome
extend
toward
the centrioles
and the
9
4
chromosomes
orient
themselves
in
a
spherical
arrangement
69
75
15
7
918
90°
58
10
14
around the centrioles. This arrangement is identical to that
120Mitotic
45Metaphase0 7Anaphase0 16Telo
32
described in Colcemid-arrested Chinese hamster fibroblasts (6).
Serial
sectioning of these cells has demonstrated the presence
a At 60 and 90 min, values include data from another recovery
of
2
normal
centriole pairs within the center of the radially
experiment in which the mitotic configurations were scored for those
times only.
arranged chromosomes (not shown). Dividing lymphocytes
arrested in C-mitosis also display large mitochondria and a
remarkably large amount of membrane located peripherally in
Table 2
the cytoplasm (Fig. 13). Much of the membrane contains
Recovery rate of CML leukocytes from treated patient
nuclear
pores and probably represents persistent nuclear
Recovery was performed on duplicate cultures. Mitotic index =1%.
envelope (Fig. 13).
CML cells blocked with Colcemid were not unlike the
(%)C-metaphase100100847680568067Metaphase00121241246Anaphase000882087Telo
configurations
Time interval
after Colcemid
normal
blocked
lymphocytes
by
light
microscope
removal
(min)0153045607590105Mitotic
phase0004812820
examination. However, the ultrastructural analysis of dividing
CML cells has yielded some interesting preliminary data, and
some unusual features were noted when CML cells were
reversed from Colcemid metaphase. Fig. 14 is an electron
micrograph of a cell taken from a culture 1 hr after removal of
Colcemid. During the recovery, a normal bipolar spindle was
formed. Figs. 15a and \5b are sections through the spindle
showing a bipolar spindle with centrioles at opposite poles.
The arrangement of mitochondria, lysosome-like bodies, and
nuclear membrane around the spindle was observed in all
sections of these cells (Figs. 13 to 16). In other sections of the
CML cells include multipolar spindles (Fig. 7), lagging cell, remnants of the Golgi apparatus can be seen (Fig. 16),
chromosomes in metaphase cells (Fig. 8), chromosome bridges
Table 3
in anaphase cells (Fig. 9), and asynchronous condensation of
Recovery rate of CML leukocytes from untreated patient
nuclei of telophase cells (Fig. 10). However, these anomalies
Recovery was performed on duplicate cultures. Mitotic index = 4%.
were seen in only 1% of dividing CML cells recovering from
Colcemid block.
Time interval
Mitotic configurations (%)
AML. One recovery experiment was attempted on three
after Colcemid
24-hr cultures of a patient with an initial diagnosis of AML.
removal (min)
C-metaphase
Metaphase
Anaphase
Telophase
The zero time specimen contained 85% C-metaphase and 15%
030609012095848578810758506437536117
telophase cells. For the remaining time periods of 30, 60, 90
and 120 min, only C-metaphase configurations could be
found. The AML cells of all 3 cultures failed to recover from
their C-metaphase configurations. In the later time periods, the
chromosomes in these cells formed condensed rings and
eventually a solid "nucleus" of chromatin was seen (Figs. 11
and 12). This patient had 78% blast cells in both peripheral
blood and bone marrow and had begun combination drug
therapy 1 day before cell culture. The drugs received included
arabinosyl cytosine, vincristine, prednisone,
and cyclophosphamide.
In both chronic and acute leukemia cultures a few anaphase
or telophase configurations were seen 2 hr after Colcemid
block and before recovery began. In the normal lymphocyte
cultures, only C-metaphase cells were seen. At the time of
addition of Colcemid, it is assumed that there are cells in
various stages of division. Apparently, normal lymphocytes are
748
Table 4
An average of the percentages of mitotic configurations seen during
the recovery of 4 CML leukocyte cultures from untreated patients
intervalremoval
Time
(%)C-metaphase9692797371Metaphase05576Anaphase128912Telophase
configurations
(min)0306090120Mitotic
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32
Mitosis in Human Leukemic Leukocytes
and groups of fibril bundles, located near the plasma
membrane, are located around the mitotic apparatus (Fig.
16).
Ultrastructural analysis of one CML patient, exhibiting the
Philadelphia
marker chromosomes
and no prior drug
treatment, revealed one cell with unusual centriole behavior. A
cell from a culture recovering from Colcemid block was
selected for electron microscope examination. By light
microscopy, the cell appeared to be in typical C-metaphase
(Fig. 17). Ultrastructurally, however, the cell had just begun
recovery. One lone centriole had migrated to the periphery of
the cell. The 3 remaining centrioles were found in the center
of the cell. Figs. 18 to 21 are adjacent sections through the 3
centrioles. The 4th centriole (Fig. 22, inset) was located at the
periphery of the cell near the section shown in Fig. 22.
Further examination of this patient's cells exhibited other
unusual features of centriole morphology. A fortuitous
longitudinal section of 1 centriole revealed the presence of 3
central vesicles (Figs. 23 and 24). Typically, only 1 central
vesicle is seen per centriole in normal cell types (7). The
significance of the multiple central vesicles in the centriole and
the frequency of such anomalies in leukemia cells remains
unknown.
Ultrastructural features of the cells from AML cultures
suggest accelerated metabolic activity. Colcemid-arrested cells
displayed strikingly large numbers of MVB, presumably in
varying stages of lysosome formation (Figs. 25 and 26). In Fig.
27, the plane of sectioning is outside the mitotic apparatus,
and it is here where lysosomes and MVB are concentrated. At
high magnification, longitudinal sections of nuclear pores can
be seen in the vast amount of membrane located throughout
these cells (Fig. 28). An interesting feature of some of the
lysosome-like bodies seen in these cells is their location in
cytoplasmic projections (Fig. 29).
DISCUSSION
The normal, C-metaphase lymphocyte reported in this study
exhibits many of the same ultrastructural features described in
other human leukocyte studies as well as other cell systems (3,
21, 22). The recovery of normal lymphocytes from the effects
of Colcemid has been shown to proceed rapidly for up to 120
min. The light micrographs taken at different time periods
during reversal illustrate the usual mitotic phases of
metaphase, anaphase, and telophase. Only typical bipolar
spindles were observed in normal cells after recovery from
Colcemid arrest. The gradual decrease in numbers of
C-metaphase cells and the subsequent increase in numbers of
telophase cells provide convincing evidence that stimulated
human lymphocytes are capable of normal recovery from
Colcemid arrest. It may be argued that the cells seen in
recovering cultures are derived from cells in G2 at the time of
Colcemid treatment. However, this seems unlikely since the
mitotic index of cultures not blocked with Colcemid is too
low to account for the increased incidence of anaphase and the
telophase configurations. Moreover, ultrastructure
studies
show the same stages of centriole migration and bipolar
spindle formation seen in other studies of cells recovering from
Colcemid block (7).
CML cells recover from Colcemid block slower than normal
cells. At 120 min after reversal, the average percentage of
telophase cells of 4 CML cultures was 11%. Correspondingly,
the percentage of C-metaphase cells decreased to an average of
only 71%. Normal lymphocyte telophase cells rose to 32% at
120 min and C-metaphose decreased to 45% at 120 min. The
reasons for the slower recovery rate in cells of CML patients is
not clear. Colcemid is known to inhibit mitosis by binding
with microtubule protein subunits (29). Recovery experiments
of 3 AML cultures suggest that these cells may be even more
sensitive to Colcemid block since they remained in the
C-metaphase configurations and no reversal was apparent.
Perhaps the Colcemid-microtubule protein complex is more
stable in cells of AML patients.
Johnson and Roberts (16) have calculated that anaphase
lasts for only 5 min in human lymphocytes; thus, in a
synchronous population, it is difficult to monitor anaphase
stages at time intervals greater than 5 min. Our data, with
samples taken at 30-min intervals, have not always shown a
gradual increase in anaphase cells during recovery. Also, the
reported percentages of metaphase cells during recovery are
believed to be low because polar view of a normal metaphase
plate is counted as a C-metaphase cell.
Rondanelli (24) has determined the mitotic rates of human
granulocytes and found that undifferentiated
blast cells
proceed through mitosis faster than the more mature
myelocytes. The duration of mitosis for myeloblasts was
reported as 43.47 to 46.25 min, and for myelocytes, 60.17 to
78.61 min; an average difference of 19.58 min (24). The
mitotic time for stimulated human lymphocytes has not been
determined precisely, but it has been reported to be between
30 and 90 min (17). The question of different mitotic time
rates of stimulated lymphocytes
or "lymphoblasts"
and
myeloblasts affecting the reversal data has not been
completely resolved. However, it seems reasonable to expect
the mitotic time of stimulated lymphocytes would not differ
significantly from that reported for immature granulocytes,
and it is felt that a comparison of their recovery rates from
Colcemid block reflects more than a simple difference in their
mitotic time rates.
Ultrastructural observations of atypical centriole structure
and behavior in cells of CML patients were striking. Evidence
for the migration of a single centriole from the remaining 3
may indicate premature maturation of daughter centrioles. In
normal mammalian mitosis, the daughter centrioles appear not
to be directly associated with spindle microtubules (6) and are
thought not to be functional in spindle microtubule formation
(6). Stubblefield (27) has observed that the frequency of
multipolar spindle increases as a function of time that Chinese
hamster cells are arrested in C-metaphase with Colcemid.
According to his interpretation, daughter centrioles mature
after prolonged arrest (3 hr or more) and upon reversal,
function as separate mitotic centers. The centriole maturation
period in CML cells may be considerably shorter than in other
cells. Further studies are necessary in order to verify this
observation. The presence of multiple central vesicles in a
centriole from a CML cell is another indication of possible
centriole abnormality. Typically, only a single central vesicle is
formed in centrioles from mammalian cells.
Our observations of centriole anomalies in the leukocytes of
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749
Manley McGill and B. R. Brinkley
CML patients are too few to permit conclusive interpretation.
Nevertheless, the importance of the ceninole in the formation
of the mitotic apparatus is well established (6, 7) and the
frequency of mitotic error in these cells may be due, in part,
to aberrant centriole structure and function. Certainly,
additional studies are warranted to confirm this view.
In 2 AML cells complete serial sectioning demonstrated
increased numbers of large lysosome-like bodies and MVB
throughout the cell and located peripheral to the mitotic
apparatus (Fig. 21 to 24). The cells, although in mitosis,
appear to be actively involved in lysosome production.
Allison et al. (l, 2) have shown that following lysosomal
activation, chromosome damage in human cells can be
demonstrated. Similar observations have been made by other
investigators (8, 23, 26). Moreover, 75% of the karyotypes of
the patient whose cells are presented in Figs. 21 to 24 were
aneuploid. It is conceivable that the increased incidence of
mitotic error in AML cells is, in some way, related to the
unusual lysosome content and activity in their cytoplasm. The
relationship of lysosome activity to the inability of these cells
to recover from Colcemid arrest is not immediately explicable
from our current knowledge of the effect of this drug on the
cell and further studies are warranted.
ACKNOWLEDGMENTS
We are indebted to Dr. J. M. Trujillo and Miss Ann Cork for thenassistance in obtaining leukemia blood specimens and providing us with
the karyotypes and medical histories of theii patients.
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CANCER
RESEARCH
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Mitosis in Human Leukemic Leukocytes
Fig. 1. Normal stimulated lymphocyte showing a C-metaphase configuration of chromosomes. Phase contrast, X 1,800.
Fig. 2. Electron micrograph of a normal stimulated lymphocyte blocked in C-metaphase. X 8,600.
Fig. 3. Light micrograph of a normal lymphocyte in metaphase configuration. Cell was taken from a culture in an early stage of recovery from
Colcemid block. Phase contrast, X 1,100.
Fig. 4. Typical anaphase configuration seen in normal lymphocyte cultures recovering from Colcemid block. Phase contrast, X 1,700.
Fig. 5. Typical telophase configuration seen in normal lymphocyte cultures recovering from Colcemid block. Phase contrast, X 1,400.
Fig. 6. Anaphase cell from a normal lymphocyte culture recovering from Colcemid block showing an anaphase bridge. Phase contrast, X 2.200.
Fig. 7. A tripolar spindle cell seen in a CML culture recovering from Colcemid block. Phase contrast, X 1,700.
Fig. 8. Metaphase cell with lagging chromosomes (Ch) in a CML culture recovering from Colcemid block. Phase contrast, X 1,100.
Fig. 9. Anaphase bridge (AB) in a cell from a CML culture recovering from Colcemid block. Phase contrast, X 1.100.
Fig. 10. Telophase cell from a CML culture recovering from Colcemid block. This cell displays an asynchronous condensation of daughter
nuclei. Phase contrast, X 1,000.
Fig. 11. Cell from an AML culture after removal of Colcemid. Cells from this culture did not recover from Colcemid block. The chromosomes in
C-metaphase formed condensed chromatin rings. Phase contrast, X 1,000.
Fig. 12. An AML cell unable to recover from Colcemid block and showing further condensation of C-metaphase chromosomes from the
condensed ring stage. Phase contrast, X 1,000.
Fig. 13. High magnification of periphery of normal dividing lymphocyte. Much of the membrane (NM) contains nuclear pores (¿VP)
and
probably represents persistent nuclear envelope. X 32,000.
Fig. 14. CML cell from a culture 1 hr after removal of Colcemid with a normal bipolar spindle formed during recovery. Note lagging
chromosome near 1 pole. X 7,800. Inset, phase-contrast micrograph of cell in Fig. 14. X 1,100.
Fig. 15. Two sections of the same cell shown in Fig. 14. a and b show a bipolar spindle with centrioles (C) at opposite poles. X 13.000.
Fig. 16. A dividing CML cell with remnants of the Golgi apparatus (G) and groups of fibrin bundles (FB) located near the plasma membrane.
X 26,000.
Fig. 17. CML cell from a culture recovering from Colcemid block. Chromosomes are arranged in a C-metaphase configuration. X 6,500. Inset,
phase-contrast micrograph of the cell in Fig. 17. X 1,100.
Figs. 18 to 21. Serial sections of the cell in Fig. 17 showing the presence of 3 centrioles. In Figs. 18 to 20, centrioles Ca and Cb are shown. In
Fig. 21, centrioles Ca and Cc can be seen and a very small part of Cb remains. A 4th ceninole was not found in this area of the cell. X 13,000.
Fig. 22. A section of the periphery of the same cell shown in Figs. 17 to 21. X 10,500. Inset, a 4th centriole located at the periphery of the cell
near the section shown in Fig. 22. X 18,000.
Fig. 23. Metaphase, CML cell recovering from Colcemid block. The longitudinal section of the mitotic apparatus revealed 1 centriole (C) with 3
central vesicles (CV). N 7,800.
Fig. 24. High magnification of the unusual centriole (C) located in the cell shown in Fig. 23. Note the 3 central vesicles (CV). X 44,000.
Fig. 25. AML cell blocked in C-metaphase. Large numbers of MVB, presumably in varying stages of lysome formation, and an increased amount
of nuclear membrane (NM) are arranged around the chromosomes (Ch). X 7,800. Inset, light micrograph of the cell in Fig. 25. Phase
contrast, X 1,100.
Fig. 26. High magnification of MVB and lysosome-like bodies (L) in an AML cell. X 11,000.
Fig. 27. AML cell blocked in C-metaphase. Plane of sectioning is outside the mitotic apparatus where MVB and lysosome-like bodies are
concentrated. X 7,800.
Fig. 28. High magnification of the periphery of an AML cell. Arrow, longitudinal sections of nuclear pores (NP) in membrane presumed to be
persistent nuclear membrane. X 24,000.
Fig. 29. AML cell in C-metaphase. Note the lysosome-like body (L) in a cytoplasmic projection at the periphery of the cell. X 10,000.
APRIL 1972
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Mitosis in Human Leukemic Leukocytes during Colcemid
Inhibition and Recovery
Manley McGill and B. R. Brinkley
Cancer Res 1972;32:746-755.
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