[CANCER
RESEARCH
28, 1773-1782, September
1968]
Differences between Diploid and Heteroploid Cultured Mammalian
Cells in Their Response to Puromycin Aminonucleoside1'2
George P. Studzinski and Kay A. O. Ellem
Department
of Pathology,
Jefferson Medical College, Philadelphia, Pennsylvania
SUMMARY
Treatment of several heteroploid cultured mammalian cell
lines with suitably low concentrations of aminonucleoside re
sulted in partial depression of ribosomal RNA synthesis,
shown by methylated albumin-Kieselguhr column chromatography of labeled nucleic acids, and a lowering of the cell UNA
content. These RNA-depleted heteroploid cell lines continued
to proliferate at control rates for several cell generations. Pro
liferation of diploid cell lines, on the other hand, was inhibited
by aminonucleoside without depression in cell RNA content.
Methylated albumin-Kieselguhr
chromatography
of nucleic
acids showed that the inhibition of ribosomal RNA synthesis
was quickly followed by the inhibition of syntheses of the other
types of RXA and of DNA. The inhibitory effects of amino
nucleoside on both heteroploid and diploid cells were prevented
by simultaneous treatment with inosine, suggesting that the
action of the inhibitor was similar in the cells studied, since
uptake of labeled aminonucleoside into the cell was not affected
by the nucleoside.
These results show that the heteroploid cell has a greater
capacity to proliferate when its rate of ribosomal RNA syn
thesis is depressed, probably because its ability to synthesize
DNA has been freed from some of the controls present in
diploid cells.
19107
culture, grow rapidly and without marked restraint by cell
to cell contact, and show a considerable proportion of abnor
malities of mitosis. Diploid cell cultures, on the other hand,
have a limited life span in culture, multiply at a slower rate
with only a rare abnormal mitotic figure, and their multiplica
tion is subject to contact inhibition by neighboring cells (6,
15, 16). Thus the growth characteristics of heteroploid cell
cultures resemble those of tumors, while diploid cell cultures
may serve as a model for nonneoplastic tissues, although it
must be admitted that some neoplastic cells appear to be dip
loid (20, 21), that karyotypic changes in cultured mammalian
cells do not always signify malignant transformation as judged
by their capacity to grow as tumors when implanted into
suitable hosts (12), and that it is difficult to find specific in
dicators of neoplastic conversion in vitro (25).
It has been shown in the first paper of this series (31) that
HeLa cells have the unexpected capacity to continue multipli
cation while their RNA synthesis is partially inhibited by
aminonucleoside of puromycin. This compound is the product
of hydrolytic cleavage of the molecule of puromycin and is
structurally analogous to adenosine (Chart 1). Unlike the
parent compound, aminonucleoside is not a specific inhibitor
of protein synthesis but inhibits the synthesis of nucleic acids.
N(CH3>2
N
INTRODUCTION
Inexorable progress towards the next cell division is the
hallmark of neoplasia. The tumor cell appears to prepare itself
for mitosis as soon as the previous division has been com
pleted, and in this process ignores the various regulatory con
trols which restrain the normal cell from incessant multiplica
tion. As was recently pointed out by Eagle (6) and Hayflick
(15), one approach to the study of mechanisms controlling
normal and abnormal growth is to compare the properties of
mammalian cell cultures which are grossly heteroploid with
those which have a strictly diploid chromosome complement.
In general, heteroploid cells can be propagated indefinitely in
NH
OCH3
NH2
Puromycin
MCCH3»,
HOCH2/0
•"
"
NH2 '
1 Supported by USPHS Grants 2RO1 CA-05402-08 and 5T01
GM-00813-06.
2 Some of these results were reported previously in a prelimi
nary form (30), and were presented at the 9th International
Cancer Congress, Tokyo, Japan, October 23-29, 1966.
Received January 17, 1968; accepted May 11, 1968.
SEPTEMBER
Ami nonucleoside
Chart 1. Comparison
and aminonucleoside.
of the structural
formulas of puromycin
1968
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1968 American Association for Cancer Research.
1773
George P. Studzinski and Kay A. 0. Ellem
When the concentration of the inhibitor is suitably low, ribosomal RXA is selectively though incompletely inhibited, al
lowing near normal synthesis of the other types of RNA and
of DXA. The RXA content of the cell is lowered, but if it does
not fall below 75% of the control value, the HeLa cell con
tinues to divide at the pretreatment rate. Higher concentrations
of aminonucleoside inhibit the synthesis of all species of nucleic
acids and after several days of treatment the rate of cell pro
liferation is retarded or division stops, but in each case the
cell shows a marked drop in its total RNA content, the cells
which cannot divide having only 50% of the RNA of control
cells. When those experiments were repeated on WI38 cells,
derived from human embryonic lung and known to be diploid,
it was found that aminonucleoside produced stoppage of cell
division in these cells without lowering their RXA content.
Several other lines of cultured mammalian cells, mostly of
human origin, were therefore tested in this way, and it was
found that a clear-cut distinction can be made between diploid
cells derived from human nonneoplastic tissues and heteroploid
cells known to be capable of producing tumors (11, 13, 26, 28)
regarding their response to aminonucleoside: only heteroploid
cells can continue to divide more than once when their RNA
synthesis is partially inhibited.
MATERIALS AND METHODS
Tissue Culture. HeLa cells used in this study were cultivated
in this laboratory for several years. Originally, they were ob
tained from the following sources: HeLa-wild from Dr. J. J.
Freed; HeLa-S3 from Dr. N. Salzman, HeLa-(-af) (Ref. 24)
from Dr. W. Henle. L cells (presumptive NCTC clone 929)
were also obtained from Dr. W. Henle; FL amnion cells were
obtained from Microbiological Associates, Bethesda, Mary
land. WI38 and WI20 cells were a gift from Dr. L. Hayflick,
and JMC1 and JMC2 cells were explanted from human em
bryonic lung and embryonic muscle respectively by Dr. R.
Love, who also established their diploid karyotype. The SRO
cell strain is a diploid line derived from adult skin fibroblasta
by Dr. S. R. Oleinick. The passage numbers of the diploid
cultures ranged from 13 to 35. Absence of mycoplasma infection
was monitored by mycoplasma culture methods (17) in the
laboratory of Dr. L. Hayflick.
All these cells were treated and handled in an identical man
ner. The cells were propagated as monolayers in plastic bottles
but were seeded into Petri dishes 72 hours before each inhibitor
experiment. The dishes were placed into a humidified C02
incubator. The cell concentration was so chosen that the con
trol cells would be in optimal logarithmic growth during the
experimental period. The medium used was in all cases Eagle's
minimal essential medium (5) supplemented with 10% calf
serum; the concentration of amino acids and vitamins was
doubled for optimal growth of diploid cells, and to ensure
uniformity of conditions this enriched medium was also used
for heteroploid cells. The medium was routinely supplemented
by kanamycin (0.167 mg per ml) or aureomycin (50 /¿gper
ml), but the antibiotics were omitted when the cells were
plated out for an experiment. During the inhibitor treatment,
the medium was changed daily on all Petri dishes. Amino
1774
nucleoside, inosine or other nucleosides, and the radioisotopes
were added to monolayers dissolved in the complete medium.
At the conclusion of each treatment period the cells were har
vested in versene (ethylenediamenetetraacetic acid in balanced
salt solution) and placed in a container surrounded by ice.
Triplicate samples were removed and diluted into 15 ml of
versene for cell enumeration with a Coulter Counter Model B
(Coulter Electronics Co., Hialeah, Fla.). The remainder of
the cell harvest was centrifuged at 0°Cat 300 X g for 10
min, the versene was removed, and the cells were washed once
with saline in the same way.
Biochemical Methods. Three aliquots of 1.5 X 10e cells each
were taken for estimation of nucleic acid content by a scaleddown version of the modified Schmidt-Thannhauser procedure
(32). The rates of nucleic acids synthesis were measured using
the dual label method previously described (8). Briefly, cytidine-5-3H and cytidine-U-14C were used as precursors, one
to label the control cells, the other to label the experimental
group. After completion of the labeling, the cells were lysed
and the nucleic acids extracted. The nucleic acids from each
group were redissolved, an aliquot taken for DNA estimation
(19), and the control and experimental samples were mixed.
The individual types of nucleic acid in the mixed sample were
resolved by methylated albumin-Kieselguhr chromatography
and the 3H/14C ratios determined for each. A second batch
was processed similarly except that the isotopes were reversed
between control and treated cultures. A calculation analogous
to taking the geometric mean of the relative rates of synthesis
for each nucleic acid in the two batches of each sample gives
a precise estimate of the relative rate of synthesis of each type
of nucleic acid in the experimental compared to the control
culture. By simultaneous chromatography of the control and
treated nucleic acids, column to column variation is eliminated,
and the effects of tailing and overlapping of fractions is min
imized by use of the 3H/14C ratios of peak fractions (8).
Effects of Inosine on the Uptake of Aminonucleoside. Cells
to be used for testing the effects of nucleosides on the uptake
of aminonucleoside were grown on coverslips in replicate ring
cultures (34). Their growth was checked by harvesting and
counting the cells. To estimate the total uptake of aminonu
cleoside, aminonucleoside-3H was presented to cells, using 4
replicate ring cultures per group, in the presence or absence
of the nucleoside. At the end of the incubation period the me
dium was removed, the steel ring wall taken off the coverslip,
and the 4 coverslips of each group passed through 4 washes
in ice cold medium, the washing time being five minutes. The
coverslips were drained after the last wash and allowed to dry.
The}' were then broken into scintillation vials, the cells dis
solved with 0.5 ml of NCS solubilizer (Nuclear Chicago), and,
after the addition of a toluene dilution of Liquifluor (Nuclear
Chicago), counted in a Nuclear Chicago scintillation spectrom
eter. Quench correction was effected using the channels ratio
method with an external standard. To provide blank levels
for isotope uptake, medium containing aminonucleoside-3H
was pipetted onto and immediately sucked off a series of ring
cultures, which were then processed as above. The counts
detectable on these cultures were subtracted from the figures
for total uptake.
CANCER RESEARCH VOL. 28
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1968 American Association for Cancer Research.
Diploid and Heteroploid Cultural Differences
Cytochemistry. Some dishes contained a small glass coverslip which was removed at the time of cell harvest, fixed in
Carnoy's fluid, and stained for 30 minutes with aqueous toluidine blue, 0.5% at pH 3 in Mcllvaine's buffer.
Materials. Tissue culture media were obtained from Grand
Island Biological Co. Aminonucleoside was purchased from
Nutritional Biochemical Co. Radioisotopes were obtained from
Nuclear Chicago. Cytidine-5-3H (specific activity 1000 me/
mmole) and uniformly labeled cytidine-14C (specific activity
233 mc/mmole) were used, with unlabeled cytidine (Schwartz
Bioresearch Inc.) present at 5 /xg/ml to provide saturating
conditions for incorporation. Uniformly labeled aminonucleoside-3H had a specific activity of 1550 mc/mmole.
RESULTS
Correlation between Cell RNA Content and the Rate of
Cell Proliferation. As has been previously shown for uncloned
HeLa cells (31), a concentration of aminonucleoside can be
found which lowers the content of RNA in FL amnion cells,
yet allows cell division to proceed for several generations at
an unimpaired rate (Chart 2). In this case such a concentra
tion of the inhibitor is 3 fig/ml, showing that this cell line is
more sensitive to aminonucleoside than HeLa cells. When the
level of aminonucleoside is increased to 6 /tg/rnl, cell prolifera
tion is retarded and eventually ceases, while cell RNA content
is markedly decreased. When a similar experiment is performed
using human embryonic lung diploid cells (Chart 3), concen
trations of 6 jig/ml or less have no apparent effect on the cell
RNA content or the rate of proliferation for the first 48 hr,
while concentrations of 12 ¿tg/mlor more arrest cell prolifera
tion after a lag of 24-48 hr. In this case the concentrations
of aminonucleoside which inhibit cell division do not affect
cell RNA content (Chart 3). It is impractical to study diploid
cell cultures for more than 72 hr, since such cultures do not
grow well when seeded at low cell concentrations.
A series of experiments such as those illustrated in Charts
2 and 3 were performed on different mammalian cell lines and
strains, and the results pertaining to the correlation between
cell RNA content and the rate of proliferation are summarized
in Table 1. Without exception, the diploid cell strains studied
in these experiments show virtually complete inhibition of cell
division after 48 hours (roughly equivalent to one generation
time of these cells) with aminonucleoside at 6 /¿g/mlor more,
and no mitotic figures can be found by microscopic examina
tion of stained cultures. The RNA content of the inhibited
diploid cells does not decrease; on the contrary, a slight in
crease is frequently seen. This contrasts with the results ob
tained with heteroploid established cell lines. Here similar
doses of aminonucleoside produce a lower cell RNA content
but allow a control rate of cell proliferation if the decrease in
cell RNA is not too severe. It should be noted that the het
eroploid lines were chosen so that they would overlap with
the diploid series with regard to generation times (JMC1 and
HeLa-wild were close in this respect) and their origin; a line
derived from adult skin was included in the diploid series
Cells per Culture
B
io-fa
50-\A\l-^\
40-/*l 3
8-Xo)
°>ÕÕ***
RNA per Cell
T]
AMS
-
ControlI
AMS
^i-ÕÕT
30-'s,l-^'
6-3"5u
ûy^'l
AMS\\Y
4-k*a
--J-7-
—
6
i
s
=
_^
I
i
20-S
**»»,
I•^
,
\
T
****~^A*j
—ï
rt6i"*~-—
a
AMS
ì'
^
—
2-"iUn.Control
24
48
72
10-n-3
24
96
Time ( hours
48
72
96
)
Chart 2. The rates of cell proliferation and RNA content in FL amnion cells treated with aminonucleoside (AMS). The points
shown represent means of 3 separate experiments, each performed in triplicate, and the standard error of the means. The numbers
in the chart represent concentration of AMS in /ig/ml. AMS at 3 Mg/ml significantly (P < 0.01) inhibited cell RNA content but
had no significant effect on the rate of cell proliferation. AMS was present from time zero.
SEPTEMBER
1968
1775
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1968 American Association for Cancer Research.
George P. Studzinski and Kay A. 0. Ellem
A
Cells per Culture
T*obx
— 4 n
B
Control
^T6
°>^fr."_"j
AMS ^\S^J
AMSX'1
7r*^»
3-<D'-I
^x
3.if-^
^ri0
'-i105
RNA per Cell
ControlffeÃ-^"''
11
T0
/
^V^
. .
•4tfi^-J:.20-j
12 AMS
2-
u1
AMS0
10--,^f12
0-^
24
48
72Time
7240-130-6
24
48
( hours
Chart 3. The rates of cell proliferation and RNA content in lung diploid cells (JMC1) treated with aminonucleoside (AMS). The
points shown represent means of 3 separate experiments, each performed in triplicate, and the standard error of the means. The
numbers in the chart represent concentration of AMS in Me/ml. Concentrations of AMS less than 6 pë/ml had no effect on cell
proliferation or RNA content during the period of study.
(SRO), and L cells, derived from a fibroblast, were among the
heteroploid lines. An incidental correlation may be seen in
Table 1: the heteroploid lines with shorter generation times
appear to be more sensitive to aminonucleoside necessitating
the use of lower concentrations of the inhibitor. This is prob
ably a reflection of the fact that a larger number of cell
divisions takes place within the experimental period.
Nucleic Acid Synthesis in Aminonucleoside-treated Cells.
Nucleic acid synthesis, as measured by incorporation of labeled
precursors, was studied in parallel with some of the experi
ments presented above, and the results are shown in Tables
2 and 3. The data shown in Table 2, obtained using radioactive
cytidine on HeLa-(-af) clone, are close to these found pre
viously on uncloned HeLa cells using 32P as precursor, which
necessitated corrections for intracellular phosphate pools (31).
In each case, in these heteroploid cells, aminonucleoside in
hibits most markedly, though incompletely, the incorporation
of the isotope into ribosomal RNA. The incorporation into
transfer RNA and 5 S RNA is less depressed, while there is
little effect on the uptake of precursors into DNA or DNAlike RNA. Similar selective inhibition by aminonucleoside of
ribosomal RNA synthesis was noted by Farnham and Dubin
(9) using L cells and sucrose gradient centrifugation for fractionation of RNA, followed by compositional analysis of the
RNA formed in the presence of the drug. On the other hand,
Table 3 shows that in diploid cells treated with aminonucleoside
for 24 hr the synthesis of all types of nucleic acids is inhibited,
with relative sparing of only DNA-like RNA. The synthesis
of DNA is depressed most of all, in complete contrast to the
situation in heteroploid cells. To determine whether this shut
down in DNA synthesis is primary or secondary, the effects
of aminonucleoside after a shorter period of action on diploid
cells was examined. In Table 3 it is evident that inhibition of
the synthesis of ribosomal RNA and the low molecular weight
1776
RNA's precedes the inhibition of DNA synthesis and that the
inhibition of these RNA types is progressive during the 24hour period* This lack of selectivity in the inhibitory effect of
aminonucleoside on DNA and RNA synthesis in diploid cells
accounts for the arrest of cell division with undiminished cell
RNA content.
Protection by Inosine against the Inhibitory Effects of
Aminonucleoside. Chart 4 shows that when inosine is admin
istered together with aminonucleoside, both heteroploid and
diploid cells continue to divide at the control rate. Deoxyinosine, adenosine, or a mixture of adenosine and guanosine do
not have this protective effect on HeLa cells, nor does deoxyinosine protect in diploid cells. Inosine not only allows the nor
mal rate of cell proliferation in the presence of moderate con
centrations of aminonucleoside, but also permits the synthesis
of the stable forms of RNA to proceed at the control rate both
in heteroploid (Chart 5) and diploid cells (Chart 6).
The protective effect of inosine against aminonucleoside is
not due to interference with the entry of the inhibitor into the
cell. The uptake of aminonucleoside-3H into the acid soluble
pools of the cell is depressed very little by simultaneous addi
tion of inosine (Table 4).
DISCUSSION
The data presented indicate a clear difference between the
group of cultures which, for want of a better indication of
their non-neoplastic properties, we shall designate as diploid,
and the cultures composed of heteroploid cells, which in many
cases have been shown to give rise to tumors when injected
into suitable animals (11-13, 26, 28). When treated with
aminonucleoside, heteroploid cells have the ability to synthesize
DNA and DNA-like RNA (which includes "messenger" RNA)
at near normal rates, although the synthesis of ribosomal RNA
CANCER RESEARCH VOL. 28
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1968 American Association for Cancer Research.
Diploid and Heteroploid Cultural Differences
Table 1
withCell
treated
Cell line or
strain and
numberDiploid
passage
lineHuman
cell
culturesWI-38
(28)"WI-38
(32)JMC-1
of the
withCell
AMSRate
/ig/ml
CellRate
us/ml AMS
of cell
of cell
RNAcontent
proliferation
mate cell
RNA
proliferation
doubting
between
between
content
at hr»13212711493103108107119109100687664787
48
time(hr;°48364848363018Cell
at hr*128113107109911021171011211037481758283«
48
48 hr«050900000011710910712095treated
and 72
hr00210000000108100991017610159Cells
48 and 72
embryoniclungHuman
(18)JMC-1
(20)JMC-1
(22)JMC-1
(30)JMC-2
embryoniclungHuman
(13)JMC-2
(14)JMC-2
(18)SRO
embryonicmuscleAdult
skinfibroblastsHuman
human
(20)SRO
(35)Established
linesHeLa-wild
heteroploid
Experiment
1Experiment
2Experiment
3Experiment
4HeLa-(-af)
ofthe carcinoma
cervixCloned
Experiment
1Experiment
2Experiment
HeLaCloned
from
3HeLa-S3
Experiment 1Origin
from HeLaApproxi
with3
treated
/ig/ml
AMSFL
cells
1Experiment
Experiment
2Experiment
amnion24816952211033857310597Cells
3Human
withL
cells
1Experiment
Experiment
treated
/ig/ml8982AMS1029757555546
subcutaneousfibroblast201
2Mouse
Correlation between cell RNA content and the rate of cell proliferation. AMS, aminonucleoside of puromycin.
a Cell doubling time was obtained by enumeration of the cell number per culture every 24 hr and plotting the data on semilogarithmic paper.
6Percent of the RNA content of untreated cells.
c Increase in the cell number per culture relative to the cell number at 48 hr, presented as percentage of similarly calculated increase
in untreated cultures during this time.
<*Passage number.
and (to a lesser extent) transfer RNA and 5 S RNA are de
pressed. As a consequence, the heteroploid cells continue to
divide for several generations in the face of decreasing content
of RNA. In diploid cells, on the other hand, aminonucleoside
produces marked inhibition of the synthesis of both DNA and
ribosomal RNA, and cell division ceases within one cell gen
eration time. In these cells RNA content is not lowered.
A more precise analysis of this difference between diploid
and heteroploid cells cannot be made at this time. The prin
cipal obstacle to fuller understanding of the molecular events
taking place in cells treated with aminonucleoside is the lack
of agreement on the mode of action of this antimetabolite,
although it is clear that unlike its parent compound, puro
mycin, aminonucleoside is not a specific inhibitor of protein
SEPTEMBER
1968
biosynthesis (2, 23). Dickie et al. (3) found that in E. coli
aminonucleoside acts as a pseudofeedback inhibitor of purine
biosynthesis, but in subsequent work (4) they withdrew an
earlier suggestion that this mechanism also explains the induc
tion of nephrotic syndrome in rat kidney (1). Farnham and
Dubin (9) suggested that aminonucleoside may act in L cells
as an analog of uncharged transfer RNA, which is thought
to inhibit ribosomal RNA synthesis in nutritionally deficient
bacteria (20, 29). In more recent studies with L cells (10),
these workers have shown that inhibition of RNA synthesis
cannot be attributed to incorporation of aminonucleoside into
RNA and that this antimetabolite does not significantly inhibit
the action of RNA polymerase. They found that adenosine
and guanosine do not prevent aminonucleoside action and were
1777
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1968 American Association for Cancer Research.
George P. Studzinski and Kay A. 0. Ellem
Table 2
fig/mlNucleic*
acidtRNA
Aminonucleoside at 12
I»
HC-labeled)3H
(control culture
(dpm)1,260
(dpm)13,073e
II
8H-labeled)3H
(control culture
(dpm)14,142
(dpm)1,113
atfig/mlsynthesis76.496.054.795.0
6
(0.14)<i
IVDNArRNA?
sRNA
12,45919,09633,314272,015
1,2762,3965,71918,496
9.767
(0.61)7.969
(0.75)5.82614.706
14,47215,52282,815262,727
9442,4371,87218,200
(0.38)6570(1.01)44.23914.436
15.331
72.9102.133.192.1
tube35°
peak
D-RNA*
80°D-RNASet
(0.30)
(0.58)
12.701 (0.86)Set
28,033»C
2,146(3H/"C)„12.707(1.00)
14,3171<C
1,127(3H/HC),10.372
13.061 (1.49)synthesis82.5
90.0Aminonucleoside
Rates of synthesis of the different nucleic acids of HeLa (-af) cells treated for 24 hours with aminonucleoside. tRNA, transfer RNA;
sRNA II, eluted peak from methylated albumin-Kieselguhr composed of tRNA and 5 S RNA ; rRNA, ribosomal RNA ; D-RNA, DNAlike RNA.
0 The actual counts (after correction to dpm) for a typical experiment are presented in this table to illustrate the method of calculat
ing the relative rates of synthesis of the nucleic acid fractions in the treated cells. The methods are dealt with in full detail elsewhere
(8). Set I is made up of a pair of cultures, one a control labeled with cytidine-U-14C for 60 min, and the other, a replicate culture
labeled with cytidine-5-3H at the same time, 24 hours after exposing it to medium containing 12 Mg/ml aminonucleoside. After labeling,
the nucleic acids were extracted and handled as described in Methods and Ref. 8. The mixed nucleic acid samples were chromatographed
together. Set II is another pair handled similarly but in which the isotopically labeled precursors were reversed.
6 The nucleic acids are listed in the order of elution from methylated albumin-Kieselguhr.
c Figures are the mean dpm of duplicate aliquots of the pooled fractions of the designated nucleic acid fraction. All samples were
counted for either 40 min or until 20,000 counts were recorded in the 14C channel. Dpm were calculated after the sample quenching had
been ascertained with an external standard. The approximate efficiencies of counting were: 3H, 30% with <0.02% falling in the 14C
channel and 14C, 65% with 15% in the 3H channel.
d The figure in parentheses is the difference between the 3H/14C of the two duplicates expressed as a percentage of their mean dpm.
e These values are the relative rates of synthesis of the nucleic acids of the treated cells as a percentage of the rate in control cells. They
were calculated using the following expression:
100 X -vi Tin" ) X (Tw" )
an<*were corrected f°rthe size of the several cultures used
on the basis of the DNA content of the aliquots mixed for chromatography (8). The use of this DNA correction factor introduces an
error related to the precision of the assay used to measure the DNA. This error affects each of the nucleic acid values within an experi
ment by the same amount; the error determines the statistical significance of the amount of the deviation of the relative rate of syn
thesis from 100%. This error does not influence the significance of the differences between the values for individual nucleic acids within
an experiment. The 95% confidence interval for the DNA correction of this experiment was ±10.6% and was similar for the other ex
periments quoted.
Errors based on the statistics of counting were estimated as the 95% confidence interval of the calculated rate percent for the sample
with the least number of counts (sRNA II) was ±0.8% of the quoted value. For the 6 Mg aminonueleoside the maximum 95% confi
dence interval was ±1.0% of the quoted value.
/3H\
/14C\
/ /"i
^!
^2
^
The formula used for calculating the 95% confidence interval of the product I j^; IX I —- 1 was a = 100 X •*
I 21
i
|
|
\ u/i
\
/n
\
\l
' 1
1
1
where Hv H2, C1( and C2 are the means of the counts recorded for the duplicate aliquots for 3H and 14C in the two series. Since the
relative rates of synthesis are the square root of the product, the 95% confidence interval for them = 100 Vl+a —100, which for small
values of a is closely approximated
a
by — X 100. This error expresses the significance of the differences between the values of the indi2
vidual nucleic acids within an experiment.
/sRNA II consists of a mixture of tRNA and 5 S RNA (e.g., Ref. 8).
* Tubes containing eluate in the rRNA zone of the chromatogram were counted individually and the tube corresponding to the peak
incorporation (Q RNA, the high molecular weight rRNA precursors) was used to calculate the rRNA values since it is least subject to
tailing and cross contamination (8).
*The tenaciously bound D-RNA was eluted in two fractions with 2% sodium dodecyl sulfate; one at 35°C,followed by the second
at 80°C.In these experiments there were no significant differences noted between their values, so that they are presented as a single
value in the other data.
1778
CANCER
RESEARCH
VOL. 28
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1968 American Association for Cancer Research.
Diploid and Heteroploid Cultural Differences
Table 3
the RNA polymerases and may inhibit the phosphorylation
of exogenously supplied purine nucleosides as well as acting
inhibitors of purine synthesis.
A number of other biochemical differences between diploid
and heteroploid cells have been described. Eagle et al. (7)
found that cultured human diploid and near-diploid cells, un
like grossly heteroploid cells, were deficient in cystathionase
activity, necessitating a more stringent nutritional requirement
for cystine by diploid cells. They suggested that enhanced
cystathionase activity was a consequence of chromosomal aber
rations. In a series of correlated spectrophotometric, interferometric, and time-lapse photographic studies, Seed (27)
showed that in cultures freshly explanted from normal tissues
(embryo human and embryo mouse), the net syntheses of
DNA, nuclear protein, and nuclear RNA are closely associated
during interphase, but in HeLa and ascites tumor cells the
synthesis of nuclear RNA and protein are dissociated to a
significant extent from DNA replication. It is also perhaps
worth noting in this general context that the modified proposal
of Warburg that neoplasia represents a shift to an anaerobic
metabolism is not yet ruled out (33), and it is argued that an
increase in glycolytic capacity may be a necessary, though not
the only, change necessary for the neoplastic transformation
(35). More recently, it was reported that there is a differential
response to inhibitors of oxidative phosphorylation on protein
synthesis in Ehrlich ascites tumor cells and normal thymocytes
(18). The authors suggested that a high energy intermediate
of oxidative phosphorylation is required for protein synthesis
in intact mammalian cells but not in tumor cells.
In all these instances the heteroploid, transformed cell has
been shown to acquire increased biosynthetic capacities and
thus to become emancipated from the limitations imposed on
its growth and division. These limitations may well be the con-
cells24
38
cells24
as feedback
hours59.357.548.856.378.5±1.8
hours80.9"833100.375.996.7±1.41
hours56.659.842.851.967.2±1.4\VI
acidtRNA
Nucleic
I«sRNA
IIDNArRNAD-RNA95%
Cl.".IMC12
Incorporation of labeled cytidine into nucleic acids of human
embryonic lung diploid cells treated with aminonucleoside. tRNA,
transfer RNA; sRNA II, eluted peak from methylated albuminKieselguhr composed of tRNA and 5 S RNA; rRNA, ribosomal
RNA; D-RNA, DNA-like RNA; C.I., confidence interval. Label
ing conditions were exactly as described in Table 2. Only one
concentration, 12 Me/ml, was used.
0 Nucleic acids as in Table 2.
6 Expressed as percent of the control value and calculated as
described in Table 2.
"The specimens (duplicate aliquots) were counted for either
20 min or 20,000 counts in the 14C channel. The 95% confidence
interval which is quoted was calculated for the samples with the
least recorded counts (sRNA II). The other fractions have smaller
95% confidence intervals.
led to believe that the drug does not act primarily as a
pseudofeedback inhibitor of purine biosynthesis. However, our
finding that inosine does protect against the inhibitory effects
of aminonucleoside calls for a réévaluation
of this possibility.
Furthermore, it is possible that the effects of aminonucleoside
are really caused by some phosphorylated derivatives of it,
rather than by the parent nucleoside (10). It is thus possible
that these active metabolites of aminonucleoside may inhibit
HeLa - Wild
B
JMCI Diploids
uu-75AMS+
Inosinex-'J
30
co
0
iS30
ControlVx/425-
D"~"\^*i*l,-1^^°
•
..^+I
AMS+
InosineControl
30
^*t^V>«*J r
AMS
15 Adenosine
Guanosine—
15
«•^
AMS^^^-'''.r..^:'^-.m
AMS30
30
Deoxyinosinet30
30
*30
AMSA0
AMS+
1 30
30
Deoxyinosine96
24
48
7230
72Time
0
( hours
24
48
)
Chart 4. Growth curves of cultures treated with aminonucleoside (AMS) together with nucleosides. The numbers in the chart
represent the concentration of the compounds in Mg/ml. Inosine protects the cells against the growth inhibitory effects of AMS, but
other nucleosides tried do not. Each point is the mean of three determinations.
SEPTEMBER
1968
1779
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1968 American Association for Cancer Research.
George P. Studzinski and Kay A. 0. Ellem
ACKNOWLEDGMENTS
trol mechanisms which regulate cell division in nonneoplastic
tissues, but the relative importance of the known differences
between normal and neoplastic cells cannot be at present
assessed. It is possible that they are all consequences of a
more basic alteration within the cell which is the crux of the
neoplastic transformation.
A
We are grateful to Miss Jeanne L. Schweitzer and Miss Agnes
T. Masse for excellent technical assistance. Mr. Robert A. Cohen
of the Jefferson Medical College and Medical Center Management
Services generously provided computer facilities, and Mr. Ken
neth Brossoie is to be thanked for writing the program.
B
Cells per Culture
•o
o
RNA per Culture
e» 400-
X
3003
U
£ 200H
(J
10012 AMS
0)
U
0
48
24
Time ( hours
72
96
)
Chart 5. Protection by inosine against the inhibitory effect of aminonucleoside (AMS) on cell proliferation and RNA accumu
lation in HeLa(-af) cell cultures. The numbers in the chart show the concentration of the compound in Me/ml. Each point is the
mean of three determinations.
Cells per Culture
2-|•00X•
B
Control
•
\/A
~+12 AMS
25 Inosine •*/,''
s\s'
60-^s**tS
80—
\+
Õ^^^^^,A
•IPJ
—
^^¿i?^*''^
40-*^ff!*f
1-^
—0) *
AMS
25 Inosine •^\S^s**"'
% \
—._._A
^
RNA per Culture
12 AMS
'i'ZZZ^Z*^
=
\Ju
/*^'*A*^n**"*^
**S^"
AMS>
2
<0-,ZQ£n-Control12
Q.JÜ~OJu
12
n-aa.
24
48
72
24
48
72
Time ( hours
Chart 6. Protection by inosine against the inhibitory effect of aminonucleoside (AMS) on cell proliferation and RNA accumu
lation in WI38 diploid cells. The numbers in the chart show the concentration of the compound in /¿g/ml. Each point is the mean
of three determinations.
1780
CANCER RESEARCH VOL. 28
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1968 American Association for Cancer Research.
Diploid and Heteroploid Cultural Differences
Table 4
Concentration
of aminonucleoside-3H
(iig/ml)0.8
30.0, Experiment
30.0, Experiment
(^Lr/inl)0
251,432
of inosinr
2.5
±190°(100)
1,320 ±259 (92)
1»
3,158 ±340 (100)
2»Concentration
1,262 ±219 (100)602,544
1,417 ±157 (99)
±191«
(78)
±169 (81)
943 ±173 (75)802,4621,172 ±154 (93)
Effect of inosine on the entry of aminon jcleoside into HeLa (-af) cells.
"Total uptake (cpm) of aminonucleoside-3H (4 /¿c/ml)into cells on cover slips, during 60 minutes at 37°C.The counts are the mean
of 4 replicates ±S.D. The values in parentheses indicate the amount of aminonucleoside taken up by the cells as a percentage
control value in the absence of inosine.
6 The average number of cells per covership in Experiment 1 was 2.99 X IO3, and in Experiment 2 was 1.2 X IO5.
cThis is the only value which is significantly different from the control at the 5% level of significance (0.02 < P < 0.05).
REFERENCES
1. Alexander, C. S., Dickie, N., and Nagasawa, H. T. Inhibition
of de novo Purine Biosynthesis as the Mechanism in Amino
nucleoside Nephrosis. 3. Lab. Clin. Med., 64: 838, 1964.
2. Allen, D. W., and Zame^nik, P. C. The Effect of Puromycin
on Rabbit Reticulocyte Ribosome. Biochim. Biophys. Acta,
65: 865-874, 1962.
3. Dickie, N., Alexander, C. S., and Nagasawa, H. T. Inhibition
of Nucleic Acid Synthesis in Escherichia Coli B by Puromycin
Aminonucleoside. Biochim. Biophys. Acta, 95: 156-169, 1965.
4. Dickie, N., Xorton, L. F., Derr, R. F., Alexander, C. S., and
Nagasawa, H. T. The Effect of Puromycin Aminonucleoside on
the Incorporation of Labeled Precursors into Rat-Kidney RNA.
Biochim. Biophys. Acta, 129: 288-293, 1966.
5. Eagle, H. Amino Acid Metabolism in Mammalian Cell Cul
tures. Science, 130: 432-137, 1959.
6. Eagle, H. Metabolic Controls in Cultured Mammalian Cells
Science, 148: 42-51, 1965.
7. Eagle, H., Washington, C., and Friedman, S. M. The Synthesis
of Homocystine,
Cystathionine,
and Cystine by Cultured
Diploid and Heteroploid Human Cells. Proc. Nati. Acad. Sci.
US., 56: 156-163, 1966.
8. Ellem, K. A. O. A Dual-Label Technique for Comparing the
Rates of Synthesis of Nucleic Acid Fractions Separated by
Methylated Albumin-Kieselguhr
Chromatography
from Cells
in Different States of Activity. Biochim. Biophys. Acta, 149:
74-87, 1967.
9. Farnham, A. E., and Dubin, D. T. Effect of Puromycin Amino
nucleoside on RNA Synthesis in L Cells. J. Mol. Biol., H:
55-62, 1965.
10. Farnham, A. E., and Dubin, D. T. Studies on Mechanism of
Action of Puromycin Aminonucleoside in L Cells. Biochim.
Biophys. Acta, 13S: 35-50, 1967.
11. Fogli, J., and Hak, K. A. Tumor Production in X-ray and
Cortisone-Treated
Rats Given Injections of Human Cells in
Continuous Cultivation. Cancer Res., IS: 692-697, 1958.
12. Foley, G. E., and Handler, A. H. Studies on the Heterotransplantability of Some Diploid Cell Lines. Exptl. Cell Res., S3:
591-594, 1964.
13. Foley, G. E., Handler, A. H., Adams, R. A., and Craig, J. M.
Assessment of Potential
Malignancy
of Cultured Cells:
Further Observations on the Differentiation of "Normal" and
"Neoplastic" Cells Maintained in Vitro by Heterotransplantation in Syrian Hamsters. Nati. Cancer Inst. Monograph, 7:
173-195, 1962.
SEPTEMBER
1968
of the
14. Hayflick, L. The Limited in vitro Lifetime of Human Diploid
Cell Strains. Exptl. Cell Res., 37: 614-636, 1965.
15. Hayflick, L. Oncogenesis in vitro. Nati. Cancer Inst. Mono
graph, ¿6:355-377, 1967.
16. Hayflick, L., and Moorhead, P. S. Serial Cultivation of Human
Diploid Cell Strains. Exptl. Cell Res., 25: 585-621, 1961.
17. Hayflick, L., and Stanbridge, E. Isolation and Identification of
Mycoplasma from Human Clinical Materials. Ann. N.Y. Acad.
Sci., 143: 608-621, 1967.
18. Jarett, L., and Kipnis, D. M. Differential Response of Protein
Synthesis in Ehrlich Ascites Tumor Cells and Normal Thymocytes to 2, 4-Dinitrophenol
and Oligomycin. Nature, 216:
714-715, 1967.
19. Rissane, J. M., and Robins, E. The Fluorometric Measure
ment of Deoxyribonucleic Acid in Animal Tissues with Special
Reference to the Central Nervous System. J. Biol. Chem.,
333: 184-188, 1958.
20. Kurland, C. G., and Maaloe, O. Regulation of Ribosomal and
Transfer RNA Synthesis. J. Mol. Biol., 4: 193-210, 1962.
21. Moore, G. E., and Sandberg, A. A. Studies of a Human Tumor
Cell Line with a Diploid Karyotype. Cancer, 17: 170-175, 1964.
22. Moorhead, P. S. Human Tumor Cell Line with a QuasiDiploid Karyotype (RPM1 2650). Exptl. Cell Res., 39: 190196, 1965.
23. Rabinovitz, M., and Fisher, J. M. A Dissociative Effect of
Puromycin on the Pathway of Protein Synthesis by Ehrlich
Ascites Tumor Cells. J. Biol. Chem., 237: 477-481, 1962.
24. Saksela, E., Fortelius, P., and Saxen, E. Chromosome Hetero
geneity of Cell Lines in vitro. Acta Pathol. Microbio!. Scand.,
51: 342-346, 1961.
25. Sanford, K. K., Barker, B. E., Woods, M. W., Parshad, R.,
and Law, L. W. Search for "Indicators" of Neoplastic Conver
sion in vitro. J. Nati. Cancer Inst , 3.9: 705-718, 1967.
26. Sanford, K. K., Earle, W. R., Sheldon, E., Schilling, E. L.,
Duehesne, E. M., Likely, G. D., and Becker, M. M. Produc
tion of Malignancy in vitro. XII. Further Transformations of
Mouse Fibroblasts to Sarcomatous Cells. J. Nati. Cancer Inst.,
;/: 351-375, 1950.
27. Seed, J. The Synthesis of DNA, RXA, and Nuclear Protein in
Normal and Tumor Strain Cells. IV. HeLa Tumor Strain Cells.
J. Cell Biol., 2S: 263-275, 1966.
28. Southam, C. M., Moore, A. E., and Rhoads, C. P. Homotrans
plantation of Human Cell Lines. Science, 1Õ5: 158-160, 1957.
29. Stent, G. S., and Brenner, S. A Genetic Locus for the Regula
tion of Ribonucleic Acid Synthesis. Proc. Nati. Acad. Sci. VS.,
47: 2005-2014, 1961.
30. Studzinski, G. P. A Difference in Growth Controlling Mech-
1781
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1968 American Association for Cancer Research.
George P. Studzinski and Kay A. 0. Ellem
anism between Diploid and Aneuploid Cell Lines. Proc. Am.
Assoc. Cancer Res., 8: 64, 1967.
31. Studzinski, G. P., and Ellem, K. A. O. Relationship between
RNA Synthesis, Cell Division, and Morphology of Mammalian Cells. I. Puromycin Aminonucleoside as an Inhibitor of
RNA Synthesis and Division in HeLa Cells. J. Cell Biol.,
39: 411-421, 1966.
32. Studzinski, G. P., and Love, R. Effects of Puromycin on the
Nucleoproteins of Hela Cells. J. Cell Biol., £2:493-503, 1964.
1782
33. Wenner, C. E. Progress in Tumor Enzymology. Advan.
Enzymol., 29: 321-389, 1967.
34. Wildy, P., Smith, C., Newton, A. A., and Dendy, P. Quantitative Cytological Studies on HeLa Cells Infected with Herpes
Virus. Virology, lo: 486-500, 1961.
35. Woods, M. W., Sanford, K. K., Burk, D., and Earle, W. R.
Glycolytic Properties of High and Low Sarcoma-producing
Lines and Clones of Mouse Tissue-Culture
Cells. J. Nati.
Cancer Inst., S3: 1079-1088, 1959.
CANCER
RESEARCH
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1968 American Association for Cancer Research.
VOL. 28
Differences between Diploid and Heteroploid Cultured
Mammalian Cells in Their Response to Puromycin
Aminonucleoside
George P. Studzinski and Kay A. O. Ellem
Cancer Res 1968;28:1773-1782.
Updated version
E-mail alerts
Reprints and
Subscriptions
Permissions
Access the most recent version of this article at:
http://cancerres.aacrjournals.org/content/28/9/1773
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 15, 2017. © 1968 American Association for Cancer Research.