(CANCER RESEARCH 42. 3798-3805,
0008-5472/82/0042-OOOOS02.00
September 1982]
Accumulation and Release of Vinblastine and Vincristine by HeLa Cells:
Light Microscopic, Cinematographic, and Biochemical Study1
Anneliese M. Lengsfeld,2 Joseph Dietrich, and Brigitte Schultze-Maurer
Institut fürMedizinische Strahlenkunde.
Universität Würzburg.Versbacher Strasse 5. D-8700 Würzburg,Federal Republic of Germany
ABSTRACT
The effect of vinblastine (VLB) and vincristine (VCR) on the
survival and proliferation of HeLa cells has been studied during
continuous and after 3-hr incubations using cell counting and
time lapse cinematography. VLB and VCR are accumulated in
the cells during drug exposure as shown with 3H-labeled drugs.
The intracellular drug concentration after a 3-hr incubation with
therapeutic doses (VLB, 0.1 ^g/ml, or VCR, 0.03 ¿ig/ml)and
thorough drug removal is 150 to 500 times higher than that in
the incubation medium. Both drugs are released from the cells
upon restoration to alkaloid-free medium reaching concentra
tions that are lethal to the cells during continuous incubation.
There is, however, a difference in the release of the two drugs.
VLB is quickly and readily released from the cells while the
release of VCR occurs slowly, since VCR is tenaciously re
tained by the cells. Both drugs are released predominantly
from living cells.
INTRODUCTION
The Vinca alkaloids VLB3 and VCR are widely used in cancer
chemotherapy. Their cell cycle phase-specific effect has been
shown in numerous in vivo and in vitro studies. These drugs
lead to mitotic arrest and subsequent necrosis and lysis not
only of cells in or shortly prior to mitosis but also of cells which
are in G2 and S phase during drug exposure while cells in d
phase continue to proliferate.
In contrast to this well-documented effect, the present stud
ies with HeLa cells have shown that 3-hr incubations with
therapeutic doses of VLB and VCR result in the death of the
cultures due to mitotic arrest, necrosis, and lysis of all cells,
i.e., also of those cells which were in d during drug exposure.
This discrepancy has led to the assumption that VLB and VCR
are accumulated in the cells and subsequently released from
the cells when these are restored to alkaloid-free medium.
Continued proliferation of the reincubated cells after additional
medium changes as well as the mitosis-arresting activity of the
medium added after drug removal have shown that this is
actually the case. Studies with 3H-labeled drugs have quantified
the accumulation
MATERIALS
and release of the drugs.
AND METHODS
Culture Conditions. HeLa cells were grown in Ham's F-10 medium
(Seromed, Munich, West Germany), supplemented with 2.5% fetal calf
serum, 12.5% horse serum, and antibiotics. The cells were normally
grown as monolayer cultures in 75-sq cm Falcon tissue culture flasks
(Falcon Plastics, Los Angeles, Calif.) and incubated under standard
conditions (5 to 6% CO2 in air saturated with water vapor, pH 7.2, at
37°).
For the experiments, 1.2 x 105 cells were seeded into 25-sq cm
Falcon flasks. For the experiments with labeled drugs, also 75-sq cm
Falcon flasks with 7.5 x 10s cells were used. After 20 to 24 hr
incubation under standard conditions, the medium was changed, and
the cells were reincubated for another 2 to 3 hr. Thereafter, the flasks
were tightly closed and transferred to an incubation chamber without
CO? or, tor time lapse observation, into a temperature-controlled
Plex
iglas cage (36.5-37.0°) surrounding the microscope.
The increase in cell number was then studied for another 16 to 24
hr prior to starting the experiments with VLB or VCR by cell counting
and/or by time lapse cinematography. Under the present experimental
conditions, the cycle time of the HeLa cells is 16 to 21 hr. The cells
grow exponentially with the same doubling time for another 2 genera
tions as shown by control cultures. At the beginning of the experiments
with VLB and VCR (about 48 hr after subcultivation), the 25-sq cm
flasks contain about 5 x 105, and the 75-sq cm ones contain 3 x 106
cells.
Synchronization. Synchronization of HeLa cells was carried out by
mitotic selection according to the procedure of Terasima and Tolmach
(10) with only slight modifications.
Cell Counts. The number of cells (interphases, mitotic cells, and
necrotic cells) was determined by repeated cell counts in 2 fields of
the flasks (marked on the bottom by a waterproof pen) each containing
60 to 150 cells at the beginning of the growth measurements 20 to 24
hr prior to drug administration. The cell number had about doubled by
the time of drug exposure. Since a distinction between arrested mitoses
and necrotic cells is difficult, only interphase cells were plotted in the
graphs as representative for surviving cells. The number of interphase
cells present after thorough drug removal has been normalized to 1; all
further cell counts are related to this cell number.
Cinematographic Procedure. Time lapse photographs were taken
with a motor-driven Bolex 16-mm camera (50-mm objective), controlled
by an automatic time lapse system (Paillard-Wild Variotimer Timer
MBF-C, Bolex-Wild Variotimer Control Unit MBF-B). The camera was
attached
contrast
distance
shielding
between
to an inverted Wild M 40 microscope equipped with phase
optics, a green filter, a heat-protecting
filter, long-working
condenser, 10x objective, and 6x eyepiece. A specimenshutter protected the cells from the microscope illumination
exposures. Eastman Plus-X negative film (Kodak; exposure
time, 0.5 sec) was used. Pictures were usually taken with 1 frame/
min. Fields with 30 to 40 cells were selected for photography.
The quantitative analysis of the time lapse films was carried out as
described previously (8) using a film viewer with a frame counter (HKS
16-mm film viewer, type LB 1600; Fa. Hünemörder,7301 Deizisau,
West Germany).
Drugs. VLB sulfate (Velbe, Lilly GmbH, Giessen, West Germany)
and VCR sulfate (Lilly) were dissolved in 0.9% NaCI solution; Ham's
medium was used for the final dilution step.
Labeled Drugs. [G- 'HJVLB sulfate (specific activity, 13.4 Ci/mmol)
and [G-3H]VCR sulfate (specific activity, 7.2 Ci/mmol)
' This work was supported by the Deutsche Forschungsgemeinschaft
(SFB
105). Dedicated to the 60th birthday of Professor Dr. K-E. Wohlfarth-Bottermann.
2 To whom requests for reprints should be addressed.
3 The abbreviations used are: VLB. vinblastine; VCR, vincristine.
Received January 11. 1982; accepted June 4. 1982.
3798
from Amersham-Buchler
GmbH, Braunschweig,
were obtained
Germany.
Experiments with Unlabeled VLB and VCR. The effect of different
drug concentrations on the survival and proliferation of HeLa cells was
studied during continuous and after 3-hr incubations: (a) continuous
CANCER
RESEARCH
Downloaded from cancerres.aacrjournals.org on July 31, 2017. © 1982 American Association for Cancer Research.
VOL. 42
Accumulation
3H activity in the pellets of the incubation
incubation with VLB (0.001, 0.0025, and 0.005 jug/ml) for up to 70 hr
and with VCR (0.0005, 0.001, and 0.0025 /tg/ml) for 70 hr; (b) 3-hr
removal and 5 washes.
In order to compare the effect of the 3H-labeled drugs with that of
142 hr. Synchronized HeLa cells were exposed to VLB (0.1 iig/m\) for
3 hr during late Gìphase and then treated as described above. In some
experiments with asynchronously growing cells, medium was addition
ally changed 5 and 10 hr after removal of the drugs. For this purpose,
the medium was collected, the cells were rinsed 3 times with Ringer's
the unlabeled drugs, survival and proliferation of the cells after 3-hr
incubation with [3H]VLB and [3H]VCR were studied by cell counting.
Furthermore, continuous incubations were carried out with low doses
of labeled drugs (0.0005, 0.001, 0.0025, and 0.005 /xg/ml for [3H]VLB; 0.00025, 0.0005, 0.001, and 0.003 ¿ig/ml for [3H]VCR) for
determining the minimum doses of the 3H-labeled drugs that are lethal
solution, and fresh medium was added.
In the case of cinematographic observation, filming started 15 to 45
min after addition or removal of the drugs and in many cases 18 to 25
hr prior to drug administration.
Experiments with [3H]VLB and [3H]VCR. Asynchronously growing
HeLa cells were exposed for 3 hr to [3H]VLB (0.1 fig/ml; diluted with
unlabeled VLB, 1:1) or to [3H]VCR (0.03 fig/ml dissolved in Ham's
during continuous
after addition,
Effect of 3-Hr Incubations with VLB and VCR on the Survival
of HeLa Cells
ME = at the
Chart 1/4 shows the effect of 3-hr incubations with VLB (0.1
and 0.025 /¿g/ml)on the survival of interphase cells. The same
is shown in Chart 1S for a 3-hr incubation with VCR (0.03 ¡ig/
ml). Although VLB and VCR were thoroughly removed after the
3-hr incubation (f = 0 in Chart 1), the number of interphase
time of removal). In the case of additional medium changes 5, 10, or
15 hr after drug removal, the cultures were washed 3 times with
Ringer's solution (R6 to R8 and R9 to R11, etc.), and fresh media were
added (M2, M3).
3H activity was measured in the 3-hr incubation medium, the different
washing solutions, in the media subsequently added as well as in the
pellets of these media containing arrested mitoses. For this purpose,
the different media and washing solutions were centrifuged for 10 min
at 2000 rpm right after collection, the supernatants were decanted,
and the 3H activity was determined by liquid scintillation counting. The
K1.0-OÃVLB\
A
incubation.
RESULTS
medium). After removal of the incubation medium (I), the cells were
washed 5 times with Ringer's solution (R1 to R5) and reincubated with
medium (MA = immediately
medium as well as of the
media removed after reincubation was determined after oxidation to
3H2O in a Tri-Carb sample oxidizer. Furthermore, the amount of
[3H]VLB and [3H]VCR retained by the cells was determined in some
samples by measuring the 3K activity of the cells trypsinized after drug
incubation with VLB (0.025 and 0.1 fig/ml); and with VCR (0.03 jig/
ml).
After 3 hr of drug exposure, cells were washed 5 to 6 times with
Ringer's solution and reincubated with alkaloid-free medium for up to
alkaloid-free
and Release of Vinca Alkaloids by HeLa Cells
cells decreases continuously after drug removal. A similar
decrease in cell number occurs during continuous incubation
with low doses of VLB (0.0025 fig/ml) or VCR (0.001 /¿g/ml).
The decrease in number of interphase cells is due to mitotic
3 hr incubation with
•'o
pg/ml\o
•0.1
(/»£\\\\\K°
0.025 pg/ml
"O
io 0.5
\°*\\\
>'>«s\\\\
°•\ü
\,
—[¿0
l
l
1
1
r
T
1
10
20
30
40
50
60
VLB
K1.0
•;
Time öfter removal
i—•
70
of VLB / hr
3hr incubation with VCR
»
\Of
(AO£.&o
0."o'5
ist\\\\
0.03 pg/ml
\\\
\
\\C\f°\•$\^
50
VCR
60
70
80
90
100
110
120
130
KO
Timeafter removalof VCR/ hr
Chart 1. Survival of HeLa cells after 3-hr incubation with VLB or VCR. The number of interphase cells present after drug removal has been normalized to 1.0. A:
•,5 experiments with a total of 7 flasks; O, 2 experiments with a total of 3 flasks. B: •,A, O, 3 experiments with a total of 11 flasks; parallel measurements of the
same experiment are represented by the same symbol. In Experiment O, cell kill caused by previous VCR exposure was not as drastic as in the other 2 experiments.
SEPTEMBER
1982
3799
Downloaded from cancerres.aacrjournals.org on July 31, 2017. © 1982 American Association for Cancer Research.
A. M. Lengsfeld et al.
N
35-
l\
cells treated during d (Chart 2) have led to the assumption
that VLB (and also VCR) might be released into the alkaloidfree medium added after drug removal as soon as the lethally
damaged cells undergo lysis. The drugs released into the
medium might then result in the same effect as a continuous
incubation with low doses of the drugs, namely, the death of all
cells.
For proving the validity of this assumption, 2 methods were
applied.
Biological Test of the Mitosis-arresting Activity of the
3hr incubationwith VLB(0.1pg/ml)
SynchronizedGì
cells
3.0
.S
2.0
.
'.5
£
k
1.0r
0.5
10
VLB
20
30
40
50
60
lime after removal of VLB / hr
70
Fresh Medium Added to the Cultures after Thorough Drug
Removal. Since it was assumed that VLB- and VCR-damaged
cells might release the drugs into the alkaloid-free medium as
soon as they undergo lysis, additional medium changes were
carried out 5 and 10 hr after drug removal, in order to remove
the arrested mitoses before they disintegrate, i.e., before they
release the drugs. These times were considered as optimal for
the removal of the arrested mitoses, since it is known from time
lapse studies that the mitotic arrest lasts about 7 to 10 hr and
the period from necrosis to lysis lasts 3 to 6 hr in VLB- and
VCR-treated HeLa cell cultures (8).4
Chart 3 shows the survival of asynchronously growing HeLa
cells after 3-hr incubation with VLB or VCR and additional
medium changes 5 and 10 hr after removal of the drugs. In
VLB-treated cultures (Chart 3A), the number of interphase cells
starts to increase shortly after the 2 medium changes. Cells
proliferate again regularly. Regular mitoses were observed
cinematographically
soon after the first additional medium
arrest and subsequent necrosis and lysis, while interphase
change 5 hr after drug removal.
death is negligible.
VCR-treated cell cultures also continue to proliferate after
This observation, the death of practically all cells following a
3-hr incubation with VLB or VCR, is in contrast to findings of additional medium changes as demonstrated in Chart 3B.
However, cells treated with VCR resume proliferation with delay
other authors and also to our own previous studies (see
"Discussion"). According to these studies, cells that are in Gì and proliferate more slowly than do VLB-treated cells. In con
phase, or at least in early G,, during exposure to VLB and VCR trast to VLB treatment, the number of interphase cells continues
are not lethally damaged by the drugs and should continue to to decrease after the first additional medium change 5 hr after
drug removal.
proliferate after drug removal.
Furthermore, the mitosis-arresting activity of the fresh me
In order to examine whether or not G, cells are also lethally
dium
added after thorough drug removal following VLB treat
damaged by VLB, synchronized HeLa cells were exposed to
ment was examined. For this purpose, the fresh medium added
VLB (0.1 (ug/ml) for 3 hr, from 5 to 8 hr after mitotic selection,
after drug removal was collected at the end of a 5-hr reincui.e., during a time period when most of the selected mitoses
bation and added to an untreated HeLa cell culture. As shown
are assumed to be in their next Gìphase. In contrast to the
in
Chart 4, this medium causes a continuous decrease in the
results with asynchronously growing cells (Chart i A), Chart 2
number of interphase cells due to mitotic arrest and necrosis
shows that cells that are in Gìduring drug exposure survive
comparable to cultures after 3-hr incubation with VLB (Chart
and continue to proliferate. That means that d cells were not
lethally damaged by the VLB treatment. The slight decrease in 1/0 or during continuous incubation with low VLB doses.
The results in Chart 3 and 4 show that VLB and VCR have
the number of interphase cells 15 to 17 hr after synchronization
indeed been released from the drug-treated cells upon resto
corresponds to an increased mitotic activity about one cycle
ration to alkaloid-free medium in a concentration sufficient to
after synchronization. However, the number of interphase cells
kill the cells. These experiments further show that VLB and
does not increase to the same extent as in synchronized
VCR must have been released predominantly from living cells
untreated controls. This is mainly due to the fact that during
and not from dead cells after lysis, since 5 hr after drug removal
the incubation period, 5 to 8 hr after mitotic selection, fastarrested mitoses did not yet disintegrate. The difference in
proliferating cells have already entered the next S phase and
resuming cell proliferation after VLB and VCR treatment and
thus become sensitive to the damaging effect of VLB. These Sadditional medium changes suggests a difference in the release
phase cells subsequently undergo mitotic arrest and lysis which
of VLB and VCR from the cells.
leads to a slower increase in the number of interphase cells
Determination of the Amount of VLB and VCR Released
than in untreated controls.
after Drug Removal Using 3H-labeled Drugs. After 3 hr incuChart 2. Survival of synchronized G, cells after 3 hr of exposure to VLB (0.1
ng/ml). HeLa cells incubated with VLB during late G, phase (5 to 8 hr after
mitotic selection) survive and continue to proliferate. Three experiments with a
total of 4 flasks; the number of interphase cells present after drug removal at
time zero normalized to 1.0.
Release of VLB and VCR
4 J. Dietrich, Kinematografische
The differences observed in the behavior of asynchronously
growing VLB-treated cells (Chart 1/4) and of synchronous HeLa
3800
und lichtmikroskopische
Untersuchungen
zur
Wirkung von Vinblastin auf HeLa Zellen. Dissertation Med. Fachbereich Würzburg
1982, in preparation.
CANCER
RESEARCH
Downloaded from cancerres.aacrjournals.org on July 31, 2017. © 1982 American Association for Cancer Research.
VOL. 42
Accumulation
and Release of Vinca Alkaloids by HeLa Cells
bation with [3H]VLB (0.1 jug/ml; Chart 5A), the 3H activity in the
Effect of medium collected from
subsequent washing solutions decreases continuously from
about 4% (R1) to about 0.5% (R5) of the total 3H activity
previously VLB treoted cells
•0.1ug/ml; ° 0.025ug/ml)
administered. When the alkaloid-free medium is added after
drug removal and the 5 washes, it contains only negligible
3.5-k3.0^KK2.5;L
3hr
VLB+
incubation with
changes*///\
additional medium
20
0
30
40
50
60
70
lime otter oddition of medium/hr
Chart 4. Survival of HeLa cells incubated with "fresh" medium collected from
VLB-treated cell cultures 5 hr after drug removal. •.exposure to "fresh" medium
^2.0:i\Kk^3
1
oi
*
•
XO/1 .
.1
.\
[\t/1
kU;0.5-i
collected from cultures treated with VLB (0.1 fig/ml); 3 experiments with a total
of 4 flasks; O, exposure to "fresh" medium collected from cultures exposed to
7o
VLB {0.025 /ig/ml); 2 experiments with a total of 3 flasks. The number of
interphase cells at the time of addition of the collected medium normalized to
1.0.
»t/
7fr
°^\\
.'. <4 /^
amounts of 3H activity (M1A). However, after 5 hr incubation,
the 3H activity in this medium has increased to about 4% (M1E).
*r
l*
ug/ml1
These 4% correspond to a 0.004-fig/ml concentration of VLB,
which is above the minimal dose lethal to the cells during
continuous incubation (VLB, 0.0025 jug/ml). Only negligible
amounts of [3H]VLB are found in the successive washing so
•0.1
VRKU
ug/mll)
o
0.025
'k'»—VLA
60»10
ti
20
30
Medium
VLB/hrchange
40
50
lutions (R6 to R11 ) and media (M2E). If the cells were incubated
in the alkaloid-free medium added after drug removal without
additional medium changes, 3 to 5% of the total [3H]VLB activity
lime aller removal of
administered
Chart 3A
were recovered
48 hr after reincubation.
This
3 hr incubation with VCR(0.03ug/ml)
+ additional medium changes
-ok'0
10
t t
VCR Medium
change
20
30
50
70
80
90
100
110
120
lime alter removal of VCR/ hr
Chart 3. Survival of HeLa cells after 3-hr incubations with VLB or VCR and additional medium changes 5 and 10 hr after drug removal. A: •,4 experiments with
a total of 5 flasks; O. 3 experiments with a total of 4 flasks. B: A, O, 2 experiments with a total of 5 flasks. The number of interphase cells present after drug removal
normalized to 1.0.
SEPTEMBER
1982
3801
Downloaded from cancerres.aacrjournals.org on July 31, 2017. © 1982 American Association for Cancer Research.
A. M. Lengsfeld et al.
100
3H-Vinblastine
„90
I
80
'eE
•o
o
=?«f
I
R1 R2 R3 R4 R5 M1A
5hr
10hr
M1ER6 R7 R8 M2A
M2ER9 RIOR11
4.5hr
100 T3
HI
90
I
80
B
4.5hr
3 H-Vincristine
C
e
o
'S
4
=
3
5hr
10hr
15hr
M1ER6 R7 R8 M2Ä
M2ER9 R10 R11M3A
M3,
01
o.
1
O
I
R1 R2 R3 R4 R5 M1A
4.5hr
4.5hr
4.5hr
Chart 5. Release of [3H]VLB and [3H]VCR from HeLa cells after 3-hr incubation with [3H]VLB (0.1 ng/ml) (A) or [3H]VCR (0.03 /ig/ml) (ß)./Abscissa, media and
washing solutions collected from HeLa cell cultures: /. | H|VLB or [3H]VCR incubation medium at the end of incubation; R1 to R11. Ringer washing solutions; Ml, M2,
M3. successive fresh media; M. . .»,fresh media immediately after addition; M. . .E, fresh media at the time of collection. Ordinate, percentage of total 3H-labeled
drugs administered that were recovered in the different media and washing solutions. B, experiments with 75-sq cm flasks containing ca. 3 x 106 cells at the time of
drug addition. 0, experiments with 25-sq cm flasks containing ca. 5 x to" cells at the time of drug addition.
corresponds also to a VLB concentration lethal during contin
uous incubation.
After 3-hr incubation with [3H]VCR (0.03 /tg/ml; Chart 56),
only the first washing solution (R1) contains a considerable
amount of 3H activity while only minimal amounts are found in
3802
the successive washing solutions. The relatively high amount
of [3H]VCR as well as [3H]VLB activity of about 3 to 4% in R1
is primarily due to the drugs remaining in the flasks after
removal of the 3-hr incubation medium as shown by the same
washing procedure with control flasks containing no cells but
CANCER
RESEARCH
Downloaded from cancerres.aacrjournals.org on July 31, 2017. © 1982 American Association for Cancer Research.
VOL. 42
Accumulation
only medium with the same [3H]VCR or [3H]VLB concentration.
The same amount of 3H activity was found in the first washing
and Release of Vinca Alkaloids by HeLa Cells
culture. Furthermore, the observation of a relatively small num
ber of cells entering mitosis during the first 5 hr after drug
removal also supports the higher cytotoxicity of [3H]VCR as
solution, R1, while R2 to R5 contained only minimal amounts,
similar to the VCR experiments. The [3H]VCR activity recovered
5 hr after reincubation with alkaloid-free medium amounts to
compared to the unlabeled drug.
2.4 to 3.3% of the administered dose (M1E) which is somewhat
less than after [3H]VLB treatment. However, the corresponding
DISCUSSION
VCR concentration of 0.0007 to 0.001 fig/ml is still in the
range of the VCR dose lethal to the cells during continuous
incubation (0.001 jig/ml). In contrast to VLB treatment, the
successive media still contain remarkable amounts of [3H]VCR
activity (M2E, M3E). If there are no additional medium changes
after reincubation with alkaloid-free medium, about 5% of the
[3H]VCR activity administered is found 23 hr after reincubation.
This corresponds to a VCR concentration of 0.0015 fig/ml and
is well above the dose which is lethal during continuous incu
bation.
These experiments clearly demonstrate not only that [3H]VLB
and [3H]VCR are released from the cells in concentrations that
kill cells if continuously applied but also that there is a differ
ence in the release of the 2 drugs from the cells. VLB is fast
and readily released, while the release of VCR occurs in a slow
and protracted fashion. This is shown by the following results:
(a) in contrast to [3H]VCR, [3H]VLB ¡scontinuously released
into the successive washing solutions following drug removal.
The [3H]VLB activity contained in the washing solutions R1 to
R5 exceeds the total amount of [3H]VLB activity remaining
within the cells after the 5 washes. (6) most of the [3H]VLB
taken up by the cells is released within the first 5 hr after drug
removal. On the contrary, only about one-half of the [3H]VCR
retained in the cells after drug removal is released within the
first 5 hr; the successive media still contain considerable
amounts of [3H]VCR. (c) after [3H]VLB incubation, the pellets
of the media contain only negligible amounts of [3H]VLB (0.1
to 2% of the supernatant), while after [3H]VCR treatment the
[3H]VCR retained in the pellets is remarkable (15 to 25%
of the supernatant), (of) when the cells were trypsinized after
thorough drug removal, nearly one-half of the [3H]VLB activity
has been released into the trypsin supernatant during the short
procedure of preparing the pellets after trypsinization, while
more than two-thirds of the [3H]VCR activity is still in the pellet
and only one-third is in the trypsin supernatant. The [3H]VLB or
[3H]VCR activity recovered in the trypsinized cells corresponds
to the amount of labeled drug released into the media after
thorough drug removal (with or without additional medium
changes).
A comparison of the effect of the 3H-labeled and the unlabeled drugs on cell proliferation and cell kill has, in the case
of VLB, shown similar cytotoxicity. After 3 hr exposure to
[3H]VLB, the increase in the number of interphase cells after
additional medium changes is in the lower range of that ob
served after incubation with unlabeled VLB. However, in the
case of VCR, the 3H-labeled drug exhibits a greater cytotoxic
effect than does the unlabeled one. After a 3-hr incubation with
[3H]VCR (0.03 jug/ml) cells do not resume proliferation after
drug removal and additional medium changes 5 and 10 hr later,
not even after a third medium change 15 hr later. This is in
contrast to the results with unlabeled VCR. In the case of
continuous incubation, the minimal lethal dose of unlabeled
VCR is 0.001 nQ/m\, while only one-half of this dose of [3H]VCR (0.0005 jug/ml) leads to the death of nearly all cells in the
SEPTEMBER1982
Uptake and Release of VLB and VCR by Drug-exposed Cells
The death of all cells in asynchronously growing HeLa cell
cultures after 3-hr incubation with VCR (0.03 jug/ml) is in
contrast to corresponding studies previously carried out cinematographically (8). These former investigations showed con
tinued proliferation of those cells which were in Gìduring the
3-hr drug exposure, while cells in S and G2 were lethally
damaged. This result was in good agreement with a number of
other in vivo and in vitro studies on the cell cycle phase-specific
effect of VCR (2, 3, 6, 7, 9). Furthermore, the death of all cells
in asynchronously growing HeLa cell cultures after 3-hr incu
bation with VLB (0.025 or 0.1 jig/ml) is in contrast to the
finding that synchronized cells exposed during d phase to the
same drug concentration survive and continue to proliferate
[Charts 1 and 2; see also Madoc-Jones and Mauro (9)]. The
present experiments have shown that the death of the cells,
also of those in Gìduring drug exposure, is due to VLB and
VCR released from the cells into the fresh medium added after
drug removal. The released drugs reach concentrations in the
fresh medium that are lethal during continuous incubation. The
experiments further showed that VLB and VCR are released
predominantly from living cells and not from cells after necrosis
and lysis, since lethal drug concentrations are reached in the
fresh medium already at a time when the arrested mitoses have
not yet started lysis.
Drug release was shown indirectly by continued proliferation
of the cells after additional medium changes (Chart 3) as well
as by testing the mitosis-arresting activity of the medium added
after drug removal (Chart 4). A direct proof of the release of
VLB and VCR was obtained by the determination of the 3H
activity in the different media and washing solutions after 3-hr
incubation with [3H]VLB and [3H]VCR. That the 3H activity
measured is indeed [3H]VLB and [3H]VCR activity was shown
by Gout ef al. (5). These authors have demonstrated chromatographically using at least 3 systems that the radioactivity elution
profile measured in blood samples as well as in the incubation
media at different time intervals up to 24 hr after application of
[3H]VLB or [3H]VCR accurately matched that of the absorbance
of VLB or VCR. The same was found by Bleyer ef al. (1 ) for
[3H]VCR recovered in the medium 1 hr after incubation of
L1210, P388, and P388/VCR cells with VCR (0.7 to 1.0 ¿ig/
ml).
Accumulation of VCR and VLB in the Cells. The mitosisarresting activity of the medium added after drug removal
(Charts 3 and 4) as well as the [3H]VLB and [3H]VCR found in
the cells just after thorough drug removal or in the media and
washing solutions subsequently added suggest that the cells
must have accumulated the drugs during the 3-hr incubation.
The extent of drug concentration in the cells was derived from
the experiments with [3H]VLB and [3H]VCR. As can be seen in
Table 1, there is no difference between the 2 drugs concerning
the accumulation by the cells after thorough drug removal.
However, there is a difference for both drugs between the
3803
Downloaded from cancerres.aacrjournals.org on July 31, 2017. © 1982 American Association for Cancer Research.
A. M. Lengsfeld et al.
Accumulation
Table 1
of VLB and VCR by HeLa cells
re
incells
tained
afterthoroughdrug
incu
ofdrug
bationmedium(ml)1010Drug
con
ad
ministered«10.3Drug
totalCell
centration(fig/ml)0.10.03Totalamount
drug)~3
no.
re
moval
(%of
con
tent/mlcells
(109cells
1ml) -
lar drug
concentra
tion com
toconcentra
pared
tion
3-hrincubationmedium167-2001
of
(fig)16.7-205-6Intracellu-
75-sq
cultureflaskVLBVCRVolumeof
cm tissue
25-sq
cm tissue
flask
VLB
VCR
5-6Drug
~5 x 105
~5 x 105
4-5
4-5
67-200
culture
0.1
0.03
0.5
0.15
accumulation by cells that were grown in 75-sq cm flasks and
by those in 25-sq cm flasks. In both cases, about 4 to 6% of
both drugs administered were retained in the cells after thor
ough drug removal. Since VLB-treated cells have already re
leased a considerable amount of the drug during the washing
procedure (R1 to R5), the accumulation of VLB by the cells
during drug exposure has been higher than that of VCR.
Under the prerequisite that 109 cells correspond to the
volume of 1 ml, the cells in the 75-sq cm flasks (3 x 106 cells)
have intracellularly accumulated the drugs 170- to 200-fold as
compared to the extracellular drug concentration in the incu
bation medium. The intracellular drug concentration of the cells
in the 25-sq cm flasks (5 x 105 cells) is even 400 to 500 times
higher than that in the 3-hr incubation medium. The reason for
this difference is not quite clear. The drug concentration of the
incubation medium is the same for both flasks. The only differ
ence is that in the 25-sq cm flasks fewer cells (5 x 10s
compared to 3 x 106) compete for a relatively large drug
amount than in the 75-sq cm flasks. Gout ef al. (5) found that,
depending on the alkaloid concentration in the incubation
medium, platelets take up 50 to 70% of the total VCR and VLB
present in the medium. The intracellular versus extracellular
drug concentration in the platelets given by these authors was
only about 2.5 for a corresponding dose of the drugs which is
much less than found in HeLa cells in the present experiments.
The bulk of the drugs accumulated inside the cell should be
bound to tubulin. However, a considerable amount seems to
be present in the cell in an unbound state. Bleyer ef al. (1)
estimated the intracellular concentration of unbound VCR to be
5.2 to 18.7 times higher than the concentration in the external
medium.
Differences in the Release of VLB and VCR from the Cells.
The present experiments reveal a marked difference in the
release of VLB and VCR from the cells. Most of the VLB is
released within the first 5 hr after drug removal and reincubation with alkaloid-free medium. Thereafter, the cells retain only
small amounts of the drug.
On the contrary, VCR is released from the cells very slowly
upon restoration to alkaloid-free medium after 3-hr incubation.
Considerable amounts of VCR are retained in the cells 5 hr
after drug removal and are continuously released into the
media subsequently added. In accordance with the slow and
protracted release of VCR is the fact that the number of
interphase cells continues to decrease after the first additional
3804
5-6~3
x 106
x 10a
40-50
12-15
400-500
400-500
medium change 5 hr after drug removal and that the VCRtreated cells recover more ¿lowly after additional medium
changes than do the VLB-treated ones (Chart 3). However, the
slow release of VCR from the cells cannot be the only reason
why VCR-treated cells resume proliferation with delay com
pared to VLB-treated cells. Although continuous incubation
with VCR is known to cause delayed progression of the cells
through the cycle (8, 9), those cells which are not lethally
damaged by the time of the second additional medium change
10 hr after removal of VCR should divide within another 10 to
15 hr and cause an increase in cell number. Therefore, it has
to be assumed that mechanisms other than the well-known
effect of VCR on spindle tubules; i.e., the effect on cytoplasmic
microtubules or other cytoplasmic receptors as the calmodulin
system (4) might be responsible for the late recovery of the
cells after VCR treatment.
The difference in the release of VLB and VCR from the cells
was also found by Gout ef al. (5). These authors showed that
WBC of the rat in vivo (mainly platelets) as well as cells from
alkaloid-sensitive rat lymphomas and L5178Y cells in vitro take
up VLB much more rapidly than VCR. When restored to alka
loid-free medium, the cells readily release VLB but retain VCR
tenaciously.
The difference in the release and retention of VLB and VCR
by the cells might have some bearing on the clinical application
of these drugs in cancer chemotherapy. It probably accounts
for the differences in oncolytic potency and side effects of the
2 drugs. In particular, the severe side effects occurring during
VCR therapy, such as neurotoxicity, could be caused by the
tenacious retention of this drug within the cells which occurs
not only in the target cells, the tumor cells, but also in other
proliferating cells as well as in cells rich in microtubules.
Effect of VCR on HeLa Cells with Different Cycle Time
The present observation that a 3-hr incubation of asynchronously growing HeLa cells with VCR (0.03 jug/ml) results in the
subsequent death of all cells in the culture does not agree with
previous cinematographic
studies (8). Those experiments
showed continued proliferation of Gìcells not lethally damaged
during the 3-hr drug exposure with the same dose. The main
difference between these experiments consists in the different
cycle time of the HeLa cells due to different experimental
conditions. In the former experiments, the mean cycle time of
CANCER
RESEARCH
Downloaded from cancerres.aacrjournals.org on July 31, 2017. © 1982 American Association for Cancer Research.
VOL. 42
Accumulation
the HeLa cells was about 40 hr (cells grown in Retri dishes),
while under the present conditions the mean cycle time is about
18 hr (cells grown in Falcon flasks).
VCR apparently affects cell populations with longer cycle
times less than those with shorter ones. There are several
reasons for this effect. The total number of cells at the time of
drug administration is smaller in slowly growing cell cultures,
which means that the total amount of VCR taken up by the cells
and subsequently released into the medium is smaller than in
fast-growing cultures. Furthermore, in cell populations with
long cycle times, relatively fewer cells are affected in the drugsensitive phases during the 3-hr incubation with VCR, regard
less of whether the S phase lengthens proportionally or remains
constant. Since in cells with longer cycle times all metabolic
processes might occur at a lower rate, not only might the
uptake and binding of the drug be different, but there might
also be less intracellular disturbance during the 3-hr drug
exposure.
Up to now, there is no knowledge of whether the uptake and
binding of VCR varies for cells in different cycle phases, in
particular between the sensitive and "nonsensitive"
cycle
phases. However, the discrepancy in the results of the former
and the present studies as well as the survival of synchronized
cells (Chart 2) suggest that cells in G, might take up VCR and
VLB to a smaller extent.
The influence of the cycle time on the extent of damage by
VCR is also shown by the differences in cell survival following
3-hr incubation with VCR in the present study (Chart IB). In
the experiment where the cells show less damage by VCR, the
doubling times of the corresponding control cultures are longer
(about 25 hr) than in the other experiments (about 16 hr).
Increased Cytotoxicity
of [3H]VCR Compared to the UnÃ-a-
beled Drug
An increased cytotoxicity
of [3H]VCR compared to the unla-
beled drug as observed in the present experiments has not
been reported by other authors using 3H-labeled VLB and/or
VCR in in vitro studies (1, 5, 11 ). On the contrary, Gout er al.
(5) state that they obtained the same results with respect to
cell survival and proliferation using 3H-labeled or unlabeled
drugs.
An increase in cytotoxicity
of [3H]VCR due to radiation dam
age cannot be excluded. Radiation damage cannot be caused
by the [3H]VCR incubation medium itself containing only 0.234
/iCi/ml, in particular because the incubation time is only 3 hr.
However, the cells accumulate VCR during drug exposure
reaching intracellular concentrations 170 to 500 times that in
the incubation medium. That means that the intracellular 3H
activity amounts to 35 to 117 niCi/ml. In the case of [3H]VLB,
the same accumulation occurs within the cells; the 3H activity
SEPTEMBER
1982
and Release of Vinca Alkaloids by HeLa Cells
within the cells is even higher, 100 to 340 ¿tCi/ml,due to the
high specific activity of [3H]VLB. However, there is almost no
effect on cell proliferation. This must be due to the difference
in drug release between VCR and VLB. VCR is released from
the cells much more slowly than VLB and therefore exerts its
damaging effect on the cells over a longer time.
The different galenic preparation of the compounds might
also be responsible for the different degree of cytotoxicity of
the 3H-labeled versus unlabeled drugs. Possibly, traces of
methanol and/or H2SO4 contained in the 3H-labeled drug prep
arations might also have some inhibiting influence on the sur
vival of the cells. On the other hand, lactose contained in
unlabeled VCR (Lilly) might have a protecting effect on the
cells.
ACKNOWLEDGMENTS
We wish to thank C. Dorsch and J. Krappel for kindly providing the HeLa cells.
We also thank E. Fromke for technical assistance.
REFERENCES
1. Bleyer, W. A., Frisby. S. A., and Oliverio, V. T. Uptake and binding of
vincristine by murine leukemia cells. Biochem. Pharmacol., 24: 633-639,
1975.
2. Camplejohn, R. S., Schultze, B., and Maurer. W. An in vivo double labelling
study of the subsequent fate of cells arrested in metaphase by vincristine in
the JB-1 mouse ascites tumour. Cell Tissue Kinet., 73. 239-250. 1980.
3. Ernst, P. Perturbation of the generation cycle of human leukemic blast cells
in vivo by vincristine. Biomedicine. 18: 484-490, 1973.
4. Gietzen, K., Wüthrich, A., Mansard, A., and Bader. H. Effects of Vinca
alkaloids on calmodulin-dependent Ca24-transport ATPase. In: W. Brade, G.
A. Nagel, and S. Seeber (eds.), Proceedings of the International Vinca
Alkaloid Symposium—Vindesine. pp. 16-26. Basel: S. Karger AG, 1981.
5. Gout, P. W.. Wijcik, L. L.. and Beer, C. T. Differences between vinblastine
and vincristine in distribution in the blood of rats and binding by platelets
and malignant cells. Eur. J. Cancer. 14: 1167-1178,
1978.
6. Jellinghaus, W.. Basler, U., Schultze. B., and Maurer, W. Double-labelling
autoradiography: a method to investigate the mechanism of action of cytostatic drugs, demonstrated with vincristine. In: W. Siegenthaler and R. Lutny
(eds.), Current Chemotherapy: Proceedings of the Tenth International Con
gress on Chemotherapy, pp. 1173-1175.
Washington, D. C.: American
Society for Microbiology, 1978.
7. Jellinghaus, W., Schultze, B., and Maurer. W. The effect of vincristine on
mouse jejunal crypt cells of differing cell age: double labelling autoradiographic studies using 3H- and "C-TdR. Cell Tissue Kinet.. 10: 147-156,
1977.
8. Lengsfeld, A. M., Schultze, B., and Maurer, W. Time-lapse studies on the
effect of vincristine on HeLa cells. Eur. J. Cancer, 17. 307-319. 1981.
9. Madoc-Jones, H., and Mauro, F. Interphase action of vinblastine and vin
cristine: differences in their lethal action through the mitotic cycle of cultured
mammalian cells. J. Cell. Physiol.. 72. 185-196, 1968.
10. Terascma, T.. and Tolmach, L. J. Growth and nucleic acid synthesis in
synchronously dividing populations of HeLa cells. Exp. Cell Res., 30: 344362. 1963.
11. Tsuruo. T., lida, H., Tsukagoshi, S., and Sakurai, Y. Overcoming of vincris
tine resistance in P 388 leukemia in vivo and in vitro through enhanced
cytotoxicity of vincristine and vinblastine by verapamil. Cancer Res., 41:
1967-1972,
1981.
3805
Downloaded from cancerres.aacrjournals.org on July 31, 2017. © 1982 American Association for Cancer Research.
Accumulation and Release of Vinblastine and Vincristine by
HeLa Cells: Light Microscopic, Cinematographic, and
Biochemical Study
Anneliese M. Lengsfeld, Joseph Dietrich and Brigitte Schultze-Maurer
Cancer Res 1982;42:3798-3805.
Updated version
E-mail alerts
Reprints and
Subscriptions
Permissions
Access the most recent version of this article at:
http://cancerres.aacrjournals.org/content/42/9/3798
Sign up to receive free email-alerts related to this article or journal.
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Department at [email protected].
To request permission to re-use all or part of this article, contact the AACR Publications
Department at [email protected].
Downloaded from cancerres.aacrjournals.org on July 31, 2017. © 1982 American Association for Cancer Research.