Download Cell Proliferation Kinetics and Drug Sensitivity of

[CANCER RESEARCH 37, 1057-1063, April 1977]
Cell Proliferation Kinetics and Drug Sensitivity of Exponential
and Stationary Populations of Cultured L1210 Cells1
Bijoy K. Bhuyan, Terry J. Fraser, and Kathy J. Day
TheUpjohnCompany,Kalamazoo,Michigan49001
SUMMARY
Drug sensitivity and cell cycle parameters of L1210 cells
in culture in exponential (10@to 4 x 10@cells/mI) and pla
teau phase (>8 x 10@cells/mI) of growth were compared.
The percentage of cells in G, , 5, and G2 + M for exponen
tial and plateau-phase cultures was 24.1 , 70.0, and 5.9, and
42.5, 45.6, and 11 .9, respectively.
These values correspond
to those reported previously for early and late L1210 mouse
leukemia cells. Cell survival (measured by cloning) after
drug exposure showed that actinomycin D, 1-(2-chloro
ethyl)-3-cyclohexyl-1 -nitnosounea, chlorozotocin , and strep
tozocin were all equally lethal to cells in both phases, and 1f3-D-anabinofunanosylCytosifle, high-specific-activity (20 Ci/
mmole) [3H]thymidine, vincnistine, daunomycin, adniamy
cm, and 5-fluonounacil were more lethal to exponential cells
than to plateau-phase cells. The percentage kill by S-phase
specific agents such as 1-/3-D-arabinOfuranosylCytOSine and
[3H]thymidine corresponded well with the percentage of
cells in S. Finally, 1,3-bis(2-chloroethyl)-1-nitrosounea was
more toxic to plateau cells than to exponential cells.
(21); Chinese hamster ovary, HAl (13); embryonic Chinese
hamster (25); human cervical carcinoma, HeLa (21); and
mouse mammary sarcoma, EMT 6 (27); and (b) cells in
suspension: mouse leukemia, L1210 (4, 29); munine masto
cytoma, P815 X2 (12). Among these cell lines, the EMT 6,
the P815 X2 and the L1210 can be maintained both in vitro
and in vivo.
We report here the cell cycle parameters and the drug
sensitivity of cultured L1210 cells in exponential and sta
tionany phases of growth. Parts of these results were re
ported previously (4). We chose the L1210 cells because of
the existence of an animal model counterpart, because it
seems to be an adequate model for human leukemia, and
because of its widespread use in screening for cancer
chemothenapeutic agents (18).
MATERIALS AND METHODS
L1210 Cell Culture. L1210 cells were maintained in cul
tune in Roswell Park Memorial Institute Medium 1634 sup
plemented with fetal calf serum (5%), NaHCO,@
(0.75 mg/mI),
penicillin (0.1 mg/mI), and stneptomycin (0.05 mg/mI) as
described previously (5). In this medium the cells grew
INTRODUCTION
exponentially with a generation time of about 12 hr. Expo
nential growth was maintained until a cell density of 4 x 10@
In 1966, Bruce et al. (8) compared the drug sensitivity of cells/mI was reached; stationary I cultures had 6 x 10@
cells/mI, and stationary II cultures had about 8 x 10@to 106
rapidly proliferating lymphoma cells to that of the hemo
cells/mi (see Chart 2). These cell counts were obtained by
poietic stem cells in the nesting state. Chemotherapeutic
agents were classified into: (a) cycle-specific agents such planting cells at 5 x 104/ml and using them after 24 (expo
as Fu,2 actinomycin D, or cyclophosphamide, which were nential), 42 (stationary I), and 72 hr (stationary II) of growth.
much more cytotoxic to cells in the proliferative state than Cells were counted in a Coulten counter.
Drug Exposure and Cell Survival. Adniamycin (NSC
in the nesting state; and (b) cycle-nonspecific agents such
as y-nadiation, which killed both cycling and resting cells. 123127), daunomycin (NSC 82151), BCNU (NSC 409962),
CCNU (NSC 79037), DCNU (NSC 178248), HU (NSC 32065),
Since then, many workers have compared the drug sensitiv
ity of cultured mammalian cells in the exponential and FU (NSC 19893), and VCR (NSC 67574) were obtained from
plateau phases of growth (1, 2, 12, 13, 21, 25, 27-29). This the Division of Cancer Treatment, National Cancer Institute,
interest is based on the similarities that exist between the Bethesda, Md. ara-C (NSC 63878) and streptozotocin (NSC
85988) were developed by The Upjohn Company. ana-C, HU,
kinetics of stationary-phase cultures and experimental tu
VCR,
adniamycin, and daunomycin were dissolved in me
mors in animals (14). The drug sensitivity of certain cell
dium. Streptozotocin was dissolved in 0.1 M potassium
types has been studied in detail: (a) cells in monolayer:
Chinese hamster ovary, CHO (2); Chinese hamster lung, V79 phosphate, pH 4. Actinomycin D was dissolved in acetone.
BCNU, CCNU, and DCNU were weighed into chilled bottles
and
were dissolved in dimethyl sulfoxide. Drugs were dis
I This study was supported in part by Contract NO1-CM-43753 with the
Division of Cancer Treatment, NIH, Department of Health, Education and
solved and then diluted in medium prior to addition to cells.
Welfare.
At the end of drug exposure, cells were centrifuged and
2 The abbreviations used are: FU, 5-fluorouracil; BCNU, 1,3-bis(2-chloro
washed to remove drug and then suspended in medium to
ethyl)-1 -nitrosourea; CCNU, 1-(2-chloroethyl)-3-cyclohexyl-1 -nitrosourea;
DCNU, @glucopyranose-(2-chIoroethyI)-nitrosoamino-carbonylamino-2-deoxy, determine cloning efficiency. Cell survival was determined
or chloroethyl-streptozotocin
or Chlorozotocin; HU, hydroxyurea; VCR,
by cloning in soft agar (16) and has been described previ
vincristine; ara-C, 1-@-D-arabinofuranosylcytosine; [3H]TdR, [3H]thymidine.
ously (5). In the calculation of percentage survivals, the
Received September 20, 1976; accepted December 29, 1976.
APRIL 1977
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1977 American Association for Cancer Research.
1057
B. K. Bhuyan et a!.
control (no drug treatment) samples were normalized to
100% survival. The coefficient of variation in determining
cell survival was about 15%, the coefficient of variation
being the standard deviation expressed as a percentage of
themean.
Glucose, Lactic Acid, and Amino Acid Analysis. The
cells were centrifuged prior to determining glucose, lactic
acid, on amino acid content of the supennatant medium. The
glucose content of the medium was determined by an enzy
matic procedure using the Glucostat provided by Worth ing
ton Biochemical Corp., Freehold, N. J. (24). Lactic acid
content was determined by an enzymatic procedure using
the test pack supplied by Calbiochem, Los Angeles, Calif.
(17). Amino acid content was determined (by Dr. A. J. Pan
cells, The Upjohn Co.) by ion-exchange chromatography on
a Spinco Model MS amino acid analyzer (22).
Determination of Macromolecule Synthesis. The incor
poration of [3H]TdR, 5-[3H]unidine, and L-[3H]valine (gener
ally labeled) into DNA, RNA, and protein, respectively, was
determined as described previously (7).
Autoradlography. The cells were prepared for autora
diography as described previously (3).
RESULTS
20
60
Growth Characteristics. The population kinetics, glu
HOURS
cose, and lactic acid concentration and the pH of the me
Chart 1. Metabolism of cells grown in a roller bottle. The results are the
dium in a roller culture are shown in Chart 1. Although the average of 2 different experiments shown as •or @,
A.
glucose was metabolized rapidly, a 0.8-mg/mi concentra
1Amino
Table
tion of glucose was remaining in the medium when expo
acid concentration
in mediumafter
L1210 celi
growth0
nential growth ceased. About a 0.9-mg/mI concentration of
hr―81
hraAmino
lactic acid was produced pen 0.9-mg/mi concentration of
acid(j.tg/ml)(@g/ml)%
used/3-Alanine24.6L-Arginine1001585L-Aspartic
glucose metabolized. The pH of the medium decreased
from an initial value of 7.3 to about 6.2 to 6.3. The amino
acid content of the medium was analyzed when the cell acid30320Asparagine-L-serine-L-3802992glutaminebL-Cystine1
density was 2 x 106cells/mI (81 hr) and at 0 hr. The results
(Table 1) indicate that: (a) up to 40% of the original concen
008218Glycine15220L-Glutamic
tration of the amino acids aspartic acid, cystine, glutamic
acid, glycine, histidine, isoleucine, lysine, proline, thneo
acid80840L-Histidine352432L-ISoleucine503040L-Leucine502158L-Lysine75
nine and valine were used during cell growth; (b) 40 to 70%
of phenylalanine and leucine were utilized; and (c) 70 to
100% of arginine,
methionine,
tynosine,
and senine-aspara
gine-glutamine were depleted from the medium.
The rate (cpm/105 cells/hr) of DNA, RNA, and protein
synthesis in the stationary cells was about 20% (range 13 to
31%) of that in the exponential cells. In order to expose the
cells to the same labeled precursor (thymidine, unidine, or
valine) concentration, both exponential and stationary cells
were centrifuged and resuspended at their original cell con
a The cell densities at 0 and 81 hr were 5 x 10@and 2 x 106/ml,
centration in fresh medium on in medium (called exponen
respectively. These samples were obtained from the experiment
tial medium) that had supported growth of exponential cells shown in Chart 1. The0-hr valueswereobtainedfrom the published
compositionof the mediumand correlatedwith the experimental0or in medium (called stationary medium) that had supported
hr values.
growth of stationary cells. For example, the rate of inconpo
b Asparagine-serine
and
glutamine
came
off
as 1 peak
from
the
nation of [3HITdR in DNA by stationary cells suspended in amino acid analyzer.
fresh, exponential, or stationary medium was 24, 30, and
C Tryptophan
21%,
respectively,
of that
of exponential
cells
suspended
the same media.
The rate of DNA synthesis can also be expressed as cpm/
10@S-phase cells. The exponential population had 78.8%
cells in S as compared to 45% in the stationary II population
1058
values
were
not
determined.
in
(Table 2). When the values were recalculated on this basis,
we found that exponential and stationary cells suspended in
fresh medium gave 5520 and 2100 DNA cpm/105 S-phase
cells. Therefore, S-phase cells in the stationary population
CANCER
RESEARCH
VOL. 37
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1977 American Association for Cancer Research.
Drug Sensitivity of Exponential and Stationary L1210
cycleGrowth
Table 2
of cells through
Distribution
cell
phaseFIow@microfluorometry'[3H]TdR
IabeledbG
(%)Exponential
(%)
G2+ M (%)
culture'
±1.7
5.9 ±0.7
S
70.1 ±2,1
Day 5 ascites―
Stationary I culture'
29.5
7.6
34.1
7.1
58.8
StationaryII culture'
42.5
11.9
45.6
44.8
Day 8 ascites―24
a The
flow-microfluorometric
mos Scientific
8.4
determinations
Laboratories,
62.9
46.878.8
were
Los Alamos,
59.2
45.0
done
by
N. M.) according
Dr.
H.
Crissman
to methods
(Los
published
Ala
before
(26).
b The
percentage
S-phase
[3H]TdR (2 Ci/mmole,
,. The
x
exponential
10@ cells/mI,
d The
values
cells
10 pCi/mI)
and
stationary
were
also
followed
I and
determined
reasons
for the
cessation
pulse
labeling
(15
mm)
with
II cultures
contained
2
x
10@,
6
x
10@,
and
7.5
to
9
respectively.
for
the
ascites
cells
were
obtained
from
were synthesizing DNA at 38% of the rate of exponential
cells.
Reasons for Onset of Stationary Phase. We investigated
2 possible
by
by autoradiography.
of exponential
Table
1 of
Hartman
et
al.
(15).
106 ___@.__@@__
..-“.
@..SURVIVAL
growth.
The 1st possibility was depletion of essential nutrients.
When medium containing stationary cells was adjusted to
pH 7.2 and then enriched by adding amino acids, vitamins,
glucose, or fetal calf serum, growth still ceased at about 2 x
106/ml. The 2nd possible reason was production of toxic
environment (such as acid pH on high lactic acid concentra
tion or toxic metabolites). Even if the pH was maintained at
7.2, growth ceased at 2 x 106cells/mI. Lactate had minimal
effect on cell growth, when added to give 400 to 800 @g/ml.
We found that partially depleted medium (which had al
ready supported cell growth to 10@cells/mI) still allowed a
fresh inoculum to grow to 8 x 105/ml with a doubling time
of 18 hr. This indicates that the accumulation of toxic me
tabolites in the partially depleted medium slowed down but
did not completely inhibitcell growth.
Our results do not clearly indicate the factor or factors
that caused the onset of stationary phase.
Viability of Cells in Exponential and Stationary Growth.
The viability, as determined by cloning efficiency, is shown
in Chart 2. Viability decreased the longer the cells stayed in
stationary phase. In this experiment the cells reached sta
tionary at about 52 hr, and by 76 hn, cell viability had
decreased to 40% of that of the exponential cells.
Stationary cells at 76 hr were diluted and replanted in
fresh medium at 5 x 104/ml. There was an initial lag phase
of 14 hr following which the cells grew with a doubling time
of 12 hr. This lag could be explained by the fact that only
40% of the 76-hr cells were viable, indicating an initial viable
inoculum of 2 x 10@cells/mi.
Distribution of Cells in Different Phases of the Cell Cy
dc. The distribution is shown in Table 2. The percentage of
S-phase cells determined by flow-micnofluorometny was sim
ilan to the values obtained by autoradiography after labeling
cells with [3H]TdR.
In these experiments cell samples were taken at 2 x 10@/
ml (mid-log), at 6 x 105/ml (beginning stationary on station
£
..-@
E
Cl)
Cl)
-I
-J
w
C.)
20
40
HOURS
Chart 2. Viability ofcells during exponential and stationary growth. Viabil
ity was determined by cloning. •,cells per ml; A, viability at different times
expressed as a percentage of the cloning efficiency of exponential cells; 0,
growth rate of a culture started from the stationary cells at 76 hr. These cells
were diluted
and planted
in fresh medium
at 5 x 10@/mI. Extrapolation
x10@
cells
inoculated)
were
viable.
of the
exponential growth curve to 0 time shows that probably 2 x 10@cells (ofthe 5
At these times the actual percentage of viable cells was 75.6
respectively, increased from 24 and 5.9% in exponential
cells to 42.5 and 11.9 in stationary II cells. This increase was
evident even at stationary I when the cell number had just
stopped increasing exponentially. The percentage of S
phase cells decreased from 70.1% in exponential cells to
45.6% in stationary II cells.
These values for L1210 cells in culture compare well with
those reported by Hartman and Dombernowsky (15) for
L1210 in vivo (Table 2). The distribution of cells in the
exponential and stationary II cultures is similar to that seen
in L1210 ascites cells obtained 5 and 8 days after injecting
10@cells.
Survival of Exponential and Stationary Cells Exposed to
Cytotoxic Agents. The survival of cells exposed to various
cytotoxic agents.is shown in Table 3. The agents can be
divided into 3 groups: (a) those that are more cytotoxic to
exponential than to stationary cells, e.g. , ara-C, HU, high
specific-activity [3H]TdR, FU, VCR, adniamycin, and dauno
mycin; (b) those that are equally cytotoxic to both groups of
cells, e.g., actinomycin D, streptozotocin, DCNU, and
± 2.6
CCNU;
any I) and at 7.5 x 105/mi (24 hr in stationary or stationary
for exponential
and
stationary
I and
51 .5 ± 2.5
II).
for
stationary II cells. The percentage of G, and G2 + M cells,
and
(C) BCNU,
which
was
more
cytotoxic
to station
ary cells than to exponential cells.
APRIL 1977
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1977 American Association for Cancer Research.
1059
B. K. Bhuyan et a!.
Table 3
Survival ofexponential
andstationary cell populationsafter cytotoxicagentsSurvival
exposure to
(%)2-hr
ExposureExponenExposure6-hr
StationaryAgent@g/mltialaStationary
IIHigh-specific-activ
0.4233.538.2ity
19.844.256.8
ExponentialStationary
I―ll@
IStationary
[3H)TdR'@ara-C530.148.547
5.237.744.4HU30023.856.2
4.348.9FU0.25
55.1cVCR0.56.326.733
0.538.2@
11.3'81.8c
28.817.9Adniamycin0.025
6.8
48.2Daunomycin0.025
0.0544.6
14.580.285.7
85.4Actinomycin
0.0569.7
16.278.5
87.8100
63
5363.4
63
4763.7
87.7
91.1
0.0969.172.1
.1
38.5
0.691
61.889.9
43.4
10
D
14.4CCNU0.5
0.016
Streptozotocin0.005
100
20065.8
1.032.3
6.549.0
,- 1.059.9
20.664.1
0.5
1.079.6
49.7
11.246.8
15.1DCNU0.5
25.3BCNU0.25
a
Exponential,
stationary
I,
b High-specific-activity
,. Cells
@
were
and
[3H]TdR
exposed
to
FU
stationary
(20
for
24
Ci/mmol)
II
populations
was
18.4
3.310@,
contained
added
to
2
give
10
1060
DCNU,
and
streptozotocin
x
6 x 10@,and 7.5 to 9 x10@ cells/mi,
respectively.
MCi/mI.
hr.
The time course of survival of cells exposed to the 5phase-specific agents, ara-C, HU, and high-specific-activity
[3H)TdR is shown in Chart 3. The results show that the
percentage of cells killed by these agents correlates well
with the percentage of S-phase cells in the population (see
Table 2). That would explain why a much higher percentage
of the exponential cells were killed than stationary cells. For
example, ara-C killed 70 and 53% of the exponential and
stationary II cells, respectively. These populations had 70
and 46% cells in S phase. These results also show that the
cells in S in the stationary population were metabolically
active, and, although they had a diminished DNA synthesis
capacity, they were still susceptible to these 5-phase-spe
cific agents. Second, the percentage of exponential cells
killed increased with time of exposure, indicating the pro
gression of non-S-phase cells into S during the exposure
period. The lower cell kill seen at 6 hr with ara-C and HU as
compared to [3H]TdR is presumably due to the accumula
tion of cells by the former agents at the G1-Sborder (5). With
the stationary population the percentage of cells killed did
not increase significantly from 2 hr to 6 hr exposure. This
suggests that very few non-S-phase cells progressed into S
during this period.
The time course of survival of cells exposed to adniamycin
and daunomycin is shown in Chart 4. The results clearly
show that at all times adniamycin and daunomycin were
much more toxic to exponential cells than to stationary
cells. For example 0.1 and 50% of the exponential and
stationary cells, respectively, survived a 6-hr exposure to
adniamycin at 0.05 @g/ml.
The cytotoxicities of the nitrosourea compounds, BCNU,
CCNU,
15.268.7
88
6451
are compared
in Table
3.
10
EXPONENTiAL
. HU,
EXPONENTiAL
Cl)
[3H] TdR, EXPONENTiAL
HOURS
Chart 3. Time course of survival of exponential and stationary II popula
tions exposed to HSA-['H]TdR, ara-C, and HU. Exponential and stationary
cell populations were taken at cell densities of 2 x 10'/ml and 7.5 to 9 x
10/mI, respectively. The cells were exposed to the agents while suspended
in the medium in which they had been growing. Solid and broken lines, cx
ponential and stationary populations, respectively. 0, •,HSA-[3H)TdR,
20 Ci/mmole, 10 @.tCi/ml; A, ara-C, 5 pg/mI; 0, •,HU, 300 @g/ml.
The greater toxicity of various doses of BCNU for station
ary cells as compared to exponential cells is shown in Chart
5. Barranco et a!. (2) and Hahn et a!. (13) also observed
CANCER RESEARCHVOL. 37
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1977 American Association for Cancer Research.
Drug Sensitivity of Exponential and Stationary L1210
found (Table 4) that there was significantly greaten toxicity
t:—-———-@
—6
DAUNOMYCIN,
STATIONARY
of BCNU for stationary cells as compared to exponential
@—::-—
——.—@
0.25 ADRIAMYCIN,
-..-.
.—
STATIONARY
____-$
0.05
cells at all concentrations of serum in the medium. In ac
condance with the finding of Hahn et a!. (13), BCNU was
more toxic in the absence than in the presence of serum.
BCNU binds to serum proteins. Also, the utilization of
serum components by cells can result in a different protein
concentration in the media supporting exponential on sta
tionary cells. In order to determine whether this effect could
modulate BCNU toxicity, the experiment shown in Table 5
was done. It is clear that stationary cells were more sensitive
than exponential cells irrespective of the medium they were
suspended in.
ADRIAMYCIN,
STATiONARY
0.025 ADRIAMYCIN,
EXPONENTiAL
-a
DISCUSSION
DAUNOMYCIN,
EXPONENTiAL
0.@
@
HOURS
Chart 4. Time course of survival of exponential and stationary II popula
tions exposed to adriamycin and daunomycin. Solid and broken lines, expo
nential and stationary populations, respectively. 0, •,
adriamycin, 0.025 @g/
ml;
A, adriamycin, 0.05 pg/mI, 0, U, daunomycin, 0.05 @g/ml.For
protocol see Chart 3.
Cell Metabolism. Our results on glucose utilization
showed that glucose was utilized rapidly even after expo
nential growth had ceased. A glucose concentration of
about 900 .tg/ml was used pen 2 x 106 cells (equivalent to
0.3 mg protein) produced. Practically all of the glucose
utilized was converted to lactic acid. Eagle et a!. (9), with
human cell cultures, and Graff and McCarty (11) with L
cells, reported that about 50 to 80% of the glucose is con
verted to lactic acid. it is quite clear that the end of expo
nential growth was not due to depletion of glucose.
Except for lysine (only 11% used up), all the amino acids
that were used less than 20% (namely, aspartic acid, cys
tine, glycine, giutamic acid, and proline) belong to “nones
sential―amino acids as identified by Levintow and Eagle
(19). These results were further corroborated (by S. L.
Kuentzel of our laboratory) when it was shown that Roswell
100
Table 4
Effect ofserum concentrationon cytotoxicitySerum
BCNU
50
\ NXPONENTIAL
inBCNUmediumSurvival(@.&g/ml)Cell
type(%)(%)0.5Exponentiai@'025.20.5Stationary―08.10.5Exponential0.543.10.5Stationar
-J
I\
N1
@4)
STATIONARY
a Exponential
and
stationary
cell
populations
contained
2
x
10@
and 7.5 to 9 x 1O@cells/mi, respectively. The cells were centrifuged
and resuspended in fresh Roswell Park Memorial Institute Medium
1634with 0, 0.5, or 5%fetal calf serum. The cells were exposedto
BCNUfor 2 hr.
Table 5
Effect of cell typeon BCNU
cytotoxicityBCNU
I.'
0.2
0.4
0.6
0.8
1.0
BCNU(
pg/mI)
typeaMedium―Survival
(%)0.5
(pg/mI)Cell
Chart 5. Survival of exponential and stationary II populations exposed to
BCNU.ForprotocolseeChart4. Theresultsaretheaverageof 3 different
experiments.
greaten toxicity of BCNU for stationary cells than for expo
nential cells.
Hahn et a!. (13) showed that the marked difference in the
sensitivity to BCNU of exponential and stationary cells de
pended on the serum concentration in the medium. We
0.5
Stationary II
Exponential
0.5
Exponential
Stationary
0.5Exponential
Stationary IIExponential
Stationary47.9
a Exponential
and
stationary
7.5 to 9 x 10@cells/mi,
ii populations
respectively.
contained
29.4
47.5
14.8
2
x
10@ and
The cells were centrifuged,
and both cells and supernatantmedium were kept. The cells were
resuspended in medium which had supported
stationary II cell populations.
either exponential
APRIL 1977
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1977 American Association for Cancer Research.
or
1061
B. K. Bhuyan et a!.
Park Memorial Institute Medium 1634 without aspartic acid,
cystine, giutamic acid, and proline could support rapid cell
growth(12 hndoublingtime), similartothatseen
in medium
containing these amino acids.
Our results did not clearly indicate the factors that caused
the onset of stationary phase. There are other possible
causes that we have not investigated. For example, Glinos
mined by microfluonometry on autonadiognaphy. These re
suits served to check the validity of our experimental tech
niques.
The greaten sensitivity of exponentially growing Li2iO
cells as compared to stationary-phase cells for the drugs we
tested has been confirmed by other workers: ana-C was
studied by Bruce et a!. (8), Wilkoff et al. (29), and Barranco
et a!.
(10)
clearly
showed
that
PO2
in medium
was
one
of the
eta!. (1). HU wasstudied by Maunoeta!. (21). However, their
factors governing the growth of cells in suspension. Thus, experiments also showed that after 6 hr exposure, more
the P02 in medium of a high-density culture was less than 10 plateau-phase V79cells were killed as compared to expo
mm Hg compared to >100 mm Hg in a low-density culture of nentially growing cells. Our results do not corroborate this
finding. Results similar to ours were also obtained by Wil
L929 cells in suspension.
Cell Viability. The viability of L1210 cells decreased nap koff et a!. (29) and Bannanco and Novak (1). Adriamycin was
idly when the cells were kept in stationary phase. Different studied by Twentyman and Bleehen (27) and Barnanco et al.
cell lines respond differently to being maintained in station
(1). FU was studied by Madoc-Jones and Bruce (20) and
any phase. For example, CHO (2), P815 (12), and EMT 6 (27) Bhuyan et a!. (6).
cellsretained
closeto 100% viability
forseveral
days inthe
For actinomycin D, VCR, and BCNU, different results
stationary phase. On the contrary, the viability of HeLa (21), have been reported by different workers using different
V79 (21),
and HA-i
cells
(14) decreased
in the stationary
systems. Twentyman and Bleehen (27) and Bruce et a!. (8)
found that actinomycin D was much more toxic to cycling
phase.
Cell Cycle Parameters. As the cells progressed from than to noncycling cells. Thatcher and Walker (25) found
exponential to stationary phase, the following changes took that confluent (i.e. , stationary) hamster embryo cells were
place. The cell number remained constant although there more sensitive to the drug than cycling cells. In our expeni
was decreased cell viability. The mitotic index decreased. ments, actinomycin D was almost equally toxic to both
The percentage of S-phase cells decreased while the pen exponentially growing and stationary cells. VCR was ne
centage of cells in G, and G2 increased. The cell size de
ported by Rossnenet a!. (23) to be more cytotoxic to station
any human lymphoid cells in culture than cycling cells.
creased.
We do not know whether the constant cell number was However, vinblastine (an analog of VCR) was found by
due to a balance between cell loss and cell proliferation or Bruce et a!. (8) to be much more cytotoxic to rapidly cycling
whether dead cells remained, without being lysed, in the lymphoma cells than to normal hemopoietic cells. In our
culture. Microscopic examination showed many small cells experiment, VCR was cleanly more active against exponen
with marked nuclear derangement in the stationary popula
tially growing cells than against stationary cells.
tion.
The variety of results obtained for BCNU in different sys
The percentage of S-phase cells determined by autora
tems has been outlined in detail by Hahn et a!. (13). Bar
diography correlated well with that determined by flow
ranco et a!. (2) found that stationary CHO cells were 1000
microfluorometry. This shows that all the cells with DNA times more sensitive than exponentially growing cells. Al
content corresponding to S phase, both in the exponential
though we did not see such a great difference in sensitivity,
and stationary populations, were synthesizing DNA. How
stationary-phase cells were consistently more sensitive than
even, the nate of DNA synthesis of the S-phase cells in were exponential cells. However, we must realize that the
stationary culture was 38% of that in exponential culture.
cell distribution in the stationary phase of L1210 is signifi
We found that, while 100% of the exponential cells were cantly different from that of CHO. Thus in stationary II
labeled by continuous exposure to [3H]TdR for 7 hr, only culture of Li210, the cells are distributed throughout the
80% of stationary
II cells were labeled
after 24 hr. Therefore
cell cycle (Table 2), whereas most of the cells reside in a
G,-Iike compartment in stationary CHO culture (2). Results
20% of the cells in stationary
culture
can be operationally
defined as noncycling and may be either truly stationary (G0) similar to ours were reported by Hageman et a!. (12) and
cells, or progressing through the cell cycle at very slow rate, Hahn et a!. (13). Twentyman and Bleehen (27) and Thatcher
and Walker (25) found that BCNU was equally cytotoxic to
or dead cells.
The distribution of cultured L12i0 cells in G,, 5, and G2+ cells in exponential and stationary phases. Our results
showed that the greaten sensitivity of the stationary cells
M in the exponential
and stationary
phases
compared
well
with those reported by Hartman and Dombernowsky (15) for depended on the cell type and not on other medium constit
uents.
5- and 8-day-old
Li2iO
ascites
(see Table
3). Hartman
and
Dombennowsky (15) reported that, according to their
The greater toxicity of BCNU for stationary cells was not
model, 96% of the G, population and 11% of the G2 popula
seen with other nitnosoureas such as CCNU and streptozo
tion in the 8-day ascites were resting (Q, and Q2) cells. tocin, the latter two being equally cytotoxic to both types of
Similar data are not yet available for the L1210 cells in cells. We do not know the reason for this difference.
culture.
The reports published cleanly show that different cell
Cytotoxicity of Drugs. ana-C and HU kill only S-phase types will respond differently with regard to the drug sensi
cells. HSA-[3H]TdR kills cells by being incorporated into the tivity of the exponential and stationary-phase cells. This
DNA and therefore
also kills only S-phase
cells.
The pen
situation exists in spite of the fact that the change in cell
centage of cells killed by these S-phase-specific agents age distribution from exponentially growing cells to station
correlated well with the percentage of S-phase cells deter
ary-phase cells is similar. Therefore, in order to use this
1062
CANCER RESEARCHVOL. 37
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1977 American Association for Cancer Research.
Drug Sensitivity of Exponential and Stationary L1210
system as a model for noncycling cells, we need to under
stand changes in cell properties (such as membrane trans
port, etc.) other than just cell-age distribution.
ACKNOWLEDGMENTS
We gratefully acknowledge the technical assistance of H. M. Wiessner, the
amino acid analyses done by Dr. A. J. Parcells, and the flow microfluorome
tric analyses done by Dr. H. Crissman (Los Alamos Scientific Laboratories).
REFERENCES
13. Hahn, G. M., Gordon, L. F., and Kurkjian. S. D. Responses of Cycling
and Noncycling Cells to 1,3-Bis(2-chloroethyl)-1-nitrosourea and BIeo
mycin. Cancer Res., 34: 2373—2379,
1974.
14.
18.
1. Barranco, S. C., and Novak, J. K. Survival Responses of Dividing and
Nondividing Mammalian Cells after Treatment with Hydroxyurea, Arabi
nosyl Cytosine, or Adriamycin. Cancer Res., 34: 1616-1628, 1974.
2. Barranco, 5. C., Novak, J. K. , and Humphrey, R. M. Response of Mam
malian Cells Following Treatment with Bleomycin and 1,3-Bis(2-chloro
ethyl)-1-nitrosourea during Plateau Phase. Cancer Res., 33:691-694,
1973.
3. Bhuyan, B. K. The Action of Streptozotocin on Mammalian Cells. Cancer
Res., 30: 2017-2023, 1970.
20.
4.
22.
Bhuyan,
B. K. Drug Sensitivity
of L1210
Cells in Exponential
and P'ateau
Phase Growth. Proc. Am. Assoc. Cancer Res., 17: 93, 1976.
5. Bhuyan, B. K., Fraser, T. J., Gray, L. G., Kuentzel, S. L., and Neil,' G. L.
Cell-Kill Kinetics of Several S-Phase-specific Drugs. Cancer Res., 33:
888-894,1973.
6. Bhuyan, B. K., Renis, H. E., and Smith, C. G. A Collagen Plate Assay for
Cytotoxic Agents. II. Biological Studies. Cancer Res.@22: 1131-1136,
19.
21.
G. M.,
and
Little,
J. B. Plateau-Phase
Cultures
of Mammalian
Johnson,
R. K., and
Goldin,
A. The
Clinical
Impact
of Screening
and
Other Experimental Tumor Studies. Cancer Treatment Rev., 2: 1-31,
1975.
Levintow, L., and Eagle, H. Biochemistry of Cultured Mammalian Cells.
Ann. Rev. Biochem., 30: 605-640, 1961.
Madoc-Jones, H., and Bruce, W. R. Sensitivity of L Cells in Exponential
and Stationary Phase to 5-fluoro-uracil. Nature, 215: 302-303, 1972.
Mauro@F., Falpo, B., Briganti, G., Elli, R., and Zupi, G. Effects of
Antineoplastic Drugs on Plateau-Phase Cultures of Mammalian Cells. II.
Bleomycin and Hydroxyurea. J. NatI. Cancer Inst., 52: 715-722, 1974.
Parcells, A. J. Determination of the Amino Acid Composition of Bovine
‘
Growth
Hormone.
Nature,
192:
971-972,
1961.
23. Rosner, F., Grunwalt, H., and Hirshaut, Y. Mechanism of Action of
Vincristine: Selective Killing of Stationary Cell Populations. Blood, 42:
1014,1973.
?@ Teller,
.
1962.
7. Bhuyan, B. K., Scheidt, L. G., and Fraser, T. J. Cell Cycle Phase Specific
ity of Antitumor Agents. Cancer Res., 32: 398-407, 1972.
8. Bruce, W. R., Meeker, B. E., and Valeriotte, F. A. Comparison of the
Sensitivity of Normal Hematopoietic and Transplanted Lymphoma Col
ony-Forming Cells to Chemotherapeutic Agents Administered in Vivo. J.
Natl. Cancer. Inst., 37: 233—245,
1966.
9. Eagle, H., Barban, S., Levy, M., and Schulze, H. 0. The Utilization of
Carbohydrates by Human Cell Cultures. J. Biol. Chem., 233: 551-558,
1958.
10. Glinos, A. 0., Vail, J. M., and Taylor, B. Density Dependent Regulation of
Growth in L Cell Suspension Cultures. Exptl. Cell Res., 78: 319-328,
1973.
11. Graft, S., and McCarty, K. S. Energy Costs of Growth in Continuous
Metazoan Cell Cultures. Cancer Res., 18: 741-746, 1958.
12. Hageman, R. F., Schenken, L. L. •
and Lesher, 5. Tumor Chemotherapy:
Efficacy Dependent on Mode of Growth. J. Natl. Cancer Inst. , 50: 467474,1973.
Hahn,
Cells. Current Topics Radiation Res. Quart., 8: 39-83, 1972.
15. Hartman, N. R., and Dombernowsky, P. Autoradiographic and Cyto
phometric Analyses of the Resting Stages of the L1210 Ascites Tumor.
Cancer Res., 34: 3296-3302, 1974.
16. Hlmmelfarb, P., Thayer, P. 5., and Martin, H. Growth of Colonies of
Leukemia L1210 in Vitro. Cancer Chemotherapy Rept., 51: 451-454,
1967.
17. Hoho@St,H. L-(+)-Lactate Determination with Lactate Dehydrogenase
and DPN. In: H. U. Bergmeyer (ed), Methods of Enzymatic Analysis, pp.
266-270. New York: Academic Press, Inc., 1963.
25.
26.
27.
28.
29.
Plasma
J. D. Direct
Glucose.
Quantitative
Abstracts
of
Colorimetric
Papers,
130th
Determination
Meeting
American
of Serum
or
Chemical
Society, September 1956, p. 69c. Washington, D. C.: American Chemical
Society News Service, 1956.
Thatcher, C. J., and Walker, I. G. Sensitivity of Confluent and Cycling
Embryonic Hamster Cells to Sulfur Mustard, 1,3-Bis(2-chloroethyl)-1nitrosourea and Actinomycin D. J. NatI. Cancer Inst. , 42: 363—368,
1969.
Tobey, R. A., and Crissman, H. A. Unique Techniques for Cell Cycle
Analysis Utilizing Mithramycin and Flow Microfluorometry. Exptl. Cell
Res., 93: 235-239, 1975.
Twentyman, P. R., and Bleehen, N. M. Changes in Sensitivity to Cyto
toxic Agents Occurring during the Life History of Monolayer Cultures of a
Mouse Tumor Cell Line. Cancer, 31: 417-423, 1975.
Van Putten, L. M., Lelieveld, P., and Kram-ldsenga, L. K. J. Cell Cycle
Specificity and Therapeutic Effectiveness of Cytostatic Agents. Cancer
Chemotherapy Rept., 56: 691-700, 1972.
Wilkoff, L. J., Dalmadge, E. A., and Lloyd, H. H. Kinetics of Effect of 1-fr
D-Arabinofuranosylcytosine, Its 5'-Palinifoyl ester, 1-$-D-Arabinofurano
syluracil and Tritiated Thymidine on the Viability of Cultured Leukemia
L1210 Cells. J. NatI. Cancer Inst., 48: 685—695,
1972.
APRIL 1977
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1977 American Association for Cancer Research.
1063
Cell Proliferation Kinetics and Drug Sensitivity of Exponential
and Stationary Populations of Cultured L1210 Cells
Bijoy K. Bhuyan, Terry J. Fraser and Kathy J. Day
Cancer Res 1977;37:1057-1063.
Updated version
E-mail alerts
Reprints and
Subscriptions
Permissions
Access the most recent version of this article at:
http://cancerres.aacrjournals.org/content/37/4/1057
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 June 16, 2017. © 1977 American Association for Cancer Research.