Download Morphological Basis for the Cytolytic Effect of

[CANCER
RESEARCH
35, 497-501,
March
1975]
Morphological Basis for the Cytolytic Effect of Vinbiastine and
Vincristine on Cultured Human Leukemic Lymphoblasts'
Awtar
Sidney
Krishan
and Emil
Frei, Ill
Farber Cancer Center and Harvard Medical School,
Boston, Massachusetts
021/5
cultured human leukemic lymphoblasts
cell line.
SUMMARY
Exposure of human leukemic lymphoblasts in suspension
cultures to low concentrations of vinblastine and vincristine
results in alterations in cell shape and leads to the formation
and release of a large number of membrane-lined
vesicles
from the cytoplasm. Separation
of these vesicles from
peripheral cytoplasm is effected through alignment and
fusion of small vacuoles. Similar vesicle formation is seen
neither in fibroblasts exposed to vinblastine nor in lympho
blasts exposed to bleomycin or adriamycin. Possible rela
tion of this phenomenon to vinblastine- and vincristine
induced cytotoxicity, spherocytosis, and thrombocytosis
is
discussed.
INTRODUCTION
Morphological alterations induced in mammalian cells by
the ymca alkaloids, vinblastine and vincristine sulfate, have
been well documented (5, 8-12). In low concentrations,
these alkaloids bind to microtubular proteins causing depo
lymerization
of spindle microtubules
and the resulting
dissolution of the mitotic spindle (5, 8, 12). In cells exposed
to high concentrations, large eosinophilic crystals (I, 10)
and ribosomal complexes (aggregates of ribosomes and
tubulin-like material) are seen in the cytoplasm ( I I).
In monolayer cultures of Earle's L-929 mouse fibroblasts
exposed to low vinblastine concentrations,
initial arrest of
MATERIALS
of the CCRF-CEM
AND METHODS
Log-phase stock cultures of human leukemic lympho
blasts, CCRF-CEM, which were initially isolated from the
peripheral blood of a pediatric leukemic patient (4), were
grown in roller bottles and nourished with Eagle's minimal
essential medium (for spinner cultures) supplemented with
10% fetal calf serum, penicillin, and streptomycin. Earle's
L-929 mouse fibroblasts and HeLa cells were grown in
monolayer
cultures and fed Eagle's minimal essential
medium supplemented with fetal calf serum and antibiotics
as above. Vinblastine sulfate (Velban) and vincristine sulfate
(Oncovin) were obtained from Eli Lilly & Co., Indianapolis,
Ind.
For light-microscopic
examination, cells were incubated
in Sykes-Moore chambers and photographed under a Nikon
inverted microscope fitted with time-lapse cinematographic
equipment.
For electron-microscopic examination, cell buttons re
trieved after light centrifugation
were fixed in 2% 0.1 M
phosphate-buffered
glutaraldehyde
(pH 7.2), postfixed in
2% buffered osmium tetraoxide, dehydrated in a graded
alcohol series, and embedded in an Epon-Araldite mixture.
Ultrathin sectionswere stained with lead citrate and uranyl
acetate and examined in a Philips 300 electron microscope.
cells in mitosis is followed by a reorganization of chromo
somes into micronuclei (8) and the formation of large,
multimicronucleated
cells. In contrast to this, exposure of
cultured human lymphoblasts of the CCRF-CEM
cell line
RESULTS
Morphological
alterations reported in the present study
were seen in both vinblastine- and vincristine sulfate-treated
pronounced
cytolysis of both the mitotic-arrested
and
CCRF-CEM
cells. However, for the sake of brevity, we
interphase cells. In the course of these observations, we have included observations
only from the vinblastine
noted pronounced alteration in the cell surface activity of treated cells.
the drug-treated lymphoblasts and the release of numerous
In CCRF-CEM
cultures exposed to vinblastine (0.1
spherical vesicles from the peripheral cytoplasm of these @zg/ml)for 24 hr, approximately 50% of the cells were dead
cells. In view of the possible connection between these or pyknotic. In contrast to this, Earle's L-929 and HeLa
observations and earlier reported vinblastine-induced
sphe
cells similarly treated had less than 10% dead or pyknotic
rocytosis (7) and thrombocytosis
(3, 6, 14), we have
cells. The pronounced cytolytic effect of these low drug
examined in the present report the structural basis for the concentrations was seen in both interphase and mitotic
pronounced cytolytic effect of vinblastine and vincristine on arrested CCRF-CEM
lymphoblasts. Although the percent
age of cells arrested in mitosis by vinblastine or vincristine
was approximately similar in L-929 and CCRF-CEM cells,
C-6516.
Received September 20. 1974: accepted November 14, 1974.
multimicronucleated
cells (formed after reconstruction
of
(4) to similar low concentrations of vinblastine results in
.
a These
MARCH
studies
were
supported
by
National
Cancer
Institute
Grant
1975
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1975 American Association for Cancer Research.
497
A . Krishan and E. Frei, III
nuclei in mitotic-arrested
CCRF-CEM
cultures.
cells) were rarely
seen in the
Effect on Cell Shape and Pseudopods. Exposure of
CCRF-CEM
lymphoblasts to low concentrations
of yin
blastine (0.01 to 0.1 .ig/ml) for 30 mm led to alterations in
both their external morphology and pseudopodal activity.
In Fig. 1, lymphoblasts from a log-phase suspension culture
are seen as more or less spherical cells with small pointed
pseudopods and occasional ruffle membranes projecting
from their leading edges (arrows). In contrast to this,
lymphoblasts exposed to vinblastine (0.01 @g/ml)for 30 mm
and shown in Fig. 2 have lost their spherical cell shape and,
instead of the numerous small pointed pseudopods, have a
single large clubfoot-like pseudopod projecting from one
end ofthe cell (arrows).
Fig. 3 shows a time-lapse sequence on the formation and
behavior of these large pseudopods
in a lymphoblast
population exposed to vinblastine (0.05 sg/ml) for approxi
mately 1 hr. The numbers on the right side of the frames
indicate mm after the addition of vinblastine to the medium.
The first (A) and the last (H) frames were photographed
after 5 1 and 70 mm of exposure to the drug. Solid arrows in
Frames A and D point to the formation and elongation of
the large foot-like cytoplasmic projection from a lympho
blast. In time-lapse movies, these foot-like cytoplasmic
projections show an unusual tendency of sticking either to
the substrate, in which case the rest of the cell rotates
around a central fixed anchor-like attachment, or to similar
processes from other cells. The latter process results in the
formation of large cell clumps normally not seen in
CCRF-CEM
lymphoblasts growing in suspension cultures.
In time-lapse Frames B to G of Fig. 3, formation of such a
large cell clump in responseto drug exposureand the final
lysis of the drug-treated cells is shown (Frames G and H).
Formation and Release of Cytoplasmic Vesicles. Besides
the above-mentioned
morphological changes, CCRF-CEM
lymphoblasts exposed to vinblastine or vincristine concen
trations of 0.01 sg/ml show numerous cytoplasmic vesicles
either in association with cells or floating freely in the
medium. A wide variation in the size range of the observed
cytoplasmic vesicles was evident; while some were as large
as 5 @imacross, many others were at the limit of microscopic
resolution.
In cell cultures mixed with trypan blue or
nigrosin black for cell viability determination, no dye
uptake was seen in the vesicles, thus suggesting the presence
of a dye-excluding cytoplasmic membrane. Free-floating
cytoplasmic vesicles were seen in culture after as short a
time as 2 hr of incubation with drug concentrations of 0.01
to 10 zg/ml. Similar cytoplasmic vesicleswere not seenin
monolayer cultures of L-929 mouse fibroblasts or HeLa
cells exposed to vinblastine or vincristine or in cultures of
CCRF-CEM
lymphoblasts exposed to bleomycin (1 to 100
@ig/ml) or adriamycin (1 to 10 zg/ml).
Fig. 4 shows a lymphoblast from a culture exposed to
vinblastine (0. 1 @ig/ml)for 6 hr. This photomicrograph was
taken from a Sykes-Moore chamber preparation and shows
numerous cytoplasmic
vesicles being released from the
peripheral
cytoplasm
of the cell. Some of the larger
cytoplasmic vesicles contain granules that may be mito
498
chondria or lipid granules. This drug-induced cytoplasmic
vesicle formation was seen in both nonmitotic interphase
cells and in cells arrested in mitosis through the stathmoki
netic action of the drug. In Fig. 5, a number of cytoplasmic
vesicles released from a mitotic-arrested
cell is seen.
In electron micrographs these drug-induced cytoplasmic
vesicles are seen as cell membrane-lined,
free-floating
portions of the ectoplasm, containing no major cellular
organelles other than ribosomes and some small vacuoles.
Fig. 6 shows a lymphoblast
from a culture exposed to
vinblastine (0.1 @tg/ml)for 24 hr. Arrows point to cytoplas
mic vesicles at different stages of their separation and
release from the peripheral cytoplasm of the lymphoblast.
One of the cytoplasmic vesicles is nearly separated from the
rest of the cell by a row of empty-looking vacuoles, whereas
a 2nd vesicle is partially separated from the rest of the
nucleus-containing
cytoplasm by a row of vacuoles on either
end.
In time-lapse movies, as seenin Fig. 3 (Frames G and H),
release of these vesicles from peripheral cytoplasm is
followed ultimately by the bursting ofthe remaining nucleus
or chromosome-containing
cytoplasm, while the cytoplas
mic vesicles float free in the medium and retain their
identity for some time.
DISCUSSION
At the outset we must distinguish between the process of
cytoplasmic vesicle formation as described in the present
study and that of cytoplasmic blebbing or zeiosis seen
occasionally in tissue culture cells (I 5). The cytoplasmic
vesicle formation,
as seen in lymphoblasts
exposed to
vinblastine, is similar to the process of platelet formation
and releasein megakaryocytes(2), whereassurfaceblebbing
of cultured cells, especially seen at anaphase, does not
involve formation and alignment of small vacuoles or result
in generation and release of small cytoplasmic vesicles.
Surface blebbing induced in interphase cells (but not in@
mitotic cells) by exposure to hypotonic solutions has been
shown to be dependent on the presence of Ca@ on the cell
surface ( I 3). A calcium-mediated gelation differential is
maintained in the cytoplasm of interphase cells, and an
inward shift of Ca@ during mitosis is responsible not only
for the breakdown of this differential but also possibly leads
to chromosomal condensation ( I 3). In view of observations
showing that vinblastine in higher concentrations
acts like
Ca@@and precipitates a number of cellular proteins as well
as calf thymus DNA (16), the possible effect of vinblastine
and vincristine on ectoplasmic gelation and cell membranes
is not unexpected. In fact, such an effect has been demon
strated in vinblastine-induced spherocytes, which resemble
those of hereditary spherocytosis in their structure, osmotic
fragility, and enhanced sodium flux rates (7). In morpholog
ical studies, Jacob et al. (7) have shown that short-term
exposure of erythrocytes from normal individuals to yin
blastine leads to the formation of numerous cup-and-bowl
shaped cells that, by fusion of their uninvaginated mem
branes, form microspherocytes. Although this process of
CANCER RESEARCH
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VOL. 35
Action of Vinca Alkaloids
“endocytosis―differs
morphologically
from that of “ex
ocytosis―
seen in the present study, the basic mechanism
responsible for the 2 processes may be similar and may
involve either 1 or both of the following effects of vinblas
tine on cells.
(a) Vinblastine reduces the structural integrity of cyto
plasm and this is reflected in drug-induced spherocytosis
and formation and release of cytoplasmic vesicles from
lymphocytes. Observations showing that higher concentra
tions of colchicine (7) will causesimilar effectsand that cells
growing attached to a substrate, as in monolayer cultures,
do not show vesicle formation support this view.
(b) The other profound effect of vinblastine may be
exercised through its action on cell membranes. The in
creased influx and efflux of sodium in erythrocytes (7) and
enhanced transport of methotrexate
in cells exposed to
vinblastine have already been reported (18). Similar mem
brane alterations are suggested by the effect of vincristine
on calcium transport and phospholipid composition of
skeletal muscle microsomes ( I7).
Present observations may also be related to vinblastine
and vincristine-induced
thrombocytosis
reported in both
human clinical cases(3, 6) and in experimental animals (14).
In rats given injections of a single dose of vincristine,
pronounced thrombocytosis
is seen on the 5th day and this
effect is not abolished in either hypersplenic or splenecto
mized rats, thereby ruling out the possibility of drug
induced platelet release from the spleen (14). In view of the
present observations,
it is possible that thrombocytosis
induced by vinblastine and vincristine may be caused by
premature release of platelets from megakaryocytes
through a mechanism similar to that described in the
present report.
On the basis of previous studies showing that the
stathmokinetic effect of vinblastine and vincristine does not
account for the selective cytocidal action of these com
pounds and that fibroblasts exposed to vinblastine and
vincristine do not show similar vesicle formation, we suggest
that this phenomenonis drug specific and may be related to
the pronounced
lymphoblasts.
cytocidal
action
of these
alkaloids
on
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Figs. I and 2. Normal and vinblastine-treated
lymphoblasts of CCRF-CEM
cell line. Arrows in Fig. I show the pointed pseudopods and ruffle
membranes of normal cells that are replaced in drug-treated cells by large foot-like projections (Fig. 2, arrows). Marker, 20 .im.
Fig. 3 shows a time-lapse sequence of lymphoblasts exposed to vinblastine (0.05 @g/ml). Numbers in the right corner of each frame indicate mm of
exposure to the drug. Arrows in Frames A to D show the formation ofa large cytoplasmic foot-like projection. Aggregation ofcells into clumps and the
subsequent release of cytoplasmic vesicles is seen in Frames D to H.
Figs. 4 and 5 show the formation and release of numerous cytoplasmic vesicles from the peripheral cytoplasm of an interphase cell (Fig. 4) and a
mitotic-arrested
cell (Fig. 5) exposed to vinblastine. Marker, 20 gzm.
Fig. 6. Arrows in this electron micrograph point to the formation of cytoplasmic vesicles from the peripheral cytoplasm of a lymphoblast exposed to
vinblastine. Rows of small vacuoles probably cause the release of the cytoplasmic vesicles by their alignment and fusion. Marker, I @.tm.
MARCH
1975
499
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CANCER RESEARCH
70
VOL. 35
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Action of y inca Alkaloids on Lymphoblasts
' ',
MARCH
-
1975
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1975 American Association for Cancer Research.
501
Morphological Basis for the Cytolytic Effect of Vinblastine and
Vincristine on Cultured Human Leukemic Lymphoblasts
Awtar Krishan and Emil Frei III
Cancer Res 1975;35:497-501.
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