Inhibition of Transcription - Resistance of African Green Monkey Kidney Cell Lines to Actinomycin D: Drug Uptake in 37 RC Cells After Persistent
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ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Feb. 1979, p. 300-312 Vol. 15, No. 2
0066-4804/79/02-0300/13$02.00/0
Resistance of African Green Monkey Kidney Cell Lines to
Actinomycin D: Drug Uptake in 37 RC Cells After Persistent
Inhibition of Transcription
ARRIGO BENEDETTO,' * ANTONIO CASSONE,2 AND CARLO DELFINI3
Centre of Virology, O.O.R.R.,' Institute of Microbiology, University of Rome,2 and Department of Cell
Biology and Immunology, Istituto Superiore di Sanita,' Rome, Italy
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Received for publication 1 December 1978
37 RC cells, a cultured line derived from African green monkey kidneys,
survived long treatments with actinomycin D (AMD; 0.1 to 0.5 ,ug/ml) under
strong inhibition of ribonucleic acid synthesis and blocking of cell division. One
aspect of the complex cellular response to this treatment was a progressive
lowering of the influx rate of AMD and, consequently, of its endocellular concen-
tration, leading to a late resurgence of transcription. Overall protein synthesis
decreased in AMD-treated cells, but more of the residual protein was exported to
the cell surface, a fact associated with the development of numerous strands of
endoplasmic reticulum and Golgi bodies in the cytoplasm. The lowering of AMD
influx during the treatment was not simply due to the decay of protein synthesis,
and there was no evidence for a carrier-mediated transport of the drug. It was
paralleled by, but seemingly not related to, modifications in cellular microtubules
and microfilaments. The rate of AMD influx was restored to levels comparable to
those of untreated cells by short exposure to ethylenediaminetetraacetic acid and
trypsin. It is concluded that the changes in plasma membrane of 37 RC cells,
creating an obstacle to the influx of AMD after long treatment with this drug,
probably consist of an accumulation and/or a different distribution of glycopro-
teins or other surface components on the cell surface.
It is well established that, whereas the pri- These cells constitute a good model for the
mary effect of actinomycin D (AMD) is exerted study of the long-term effects of AMD on cellu-
on overall transcription, not all major ribonu- lar physiology. It is clear that at suitable sub-
cleic acid (RNA) classes are inhibited to the maximal doses of AMD an "unbalanced" tran-
same extent. Indeed, large cistrons are more scription occurs in AGMK cells (2), but the
sensitive to AMD than are smaller ones (25), the mechanism by which the natural AMD resist-
inhibition increasing in the order transfer RNA ance of these cells follows from this unbalance
-+ messenger RNA -- ribosomal RNA (24, 25). has never been explained.
In the large class of messenger RNAs, AMD To obtain some insight into this problem and
sensitivity varies according to the size of the information on membrane changes in 37 RC
nuclear precursors (25). cells after long exposure to AMD, we examined
37 RC, Vero, and other epithelial-like cell more closely the possibility that continuous
lines, derived from African green monkey kidney presence of the drug, at concentrations that un-
(AGMK) cells, are known for their natural re- balance the transcription, eventually affects the
sistance to AMD as compared to the majority of uptake of AMD itself. Thus we found that: (i)
cell cultures commonly employed (3, 27, 35). after AMD exposure, the influx rate of the drug
Their resistance is expressed by a lower cytotox- was progressively reduced; (ii) when this reduc-
icity (27, 35) and rapid recovery of RNA synthe- tion reached a critical value RNA synthesis in-
sis after maximal AMD suppression (4, 28, 29), creased; (iii) this reduction in AMD influx is
a property retained even after repeated AMD probably mediated by factors from the cell sur-
blocks (4). The RNA classes recover in the order face coat which are removable by ethylenedia-
transfer RNA -* messenger RNA -* ribosomal minetetraacetic acid (EDTA)-trypsin solutions.
RNA (2), which is the reverse of inhibition,
suggesting that for each RNA class the sensitiv- MATERIALS AND METHODS
ity to and recovery from AMD obey the same Cell cultures and AMD treatment. The 37 RC
general rules. cell line, used throughout this study, was maintained
300VOL. 15, 1979 AMD RESISTANCE OF 37 RC CELLS 301
as monolayers on glass bottles in Eagle medium con- and 0.5% SDS, were dissolved in Bray solution, and
taining 5% fetal calf serum (MEM) as described else- the radioactivity was measured (incorporation into
where (2). The cultures were routinely checked for acid-precipitable material).
mycoplasma contamination by the method of Barile RNA extraction and purification. 37 RC mono-
and Schimke (1) and by electron microscope obser- layers in Kolle bottles were exposed to [3H]uridine (5
vations (see below). All experiments on kinetics of ,uCi/ml) in serum-free MEM in the absence and pres-
[3H]AMD binding were carried out with cells trans- ence of different AMD concentrations. After 30 min of
ferred into Linbro plates, each containing 24 small labeling, monolayers were quickly chilled by adding
wells (area = 1.8 cm2), and seeded at the density of 10' frozen crushed MEM. The cells were scraped from the
cells per cm2 in a total volume of 1 ml. The mediutn bottles with a rubber policeman and collected by cen-
(with or without AMD) was changed daily. The cul- trifugation at 800 x g for 5 min at 2 to 4°C. The pellet
tures were maintained in a CO2 incubator at 37°C. was then suspended in RSB buffer [10 mM NaCl;
Cells were routinely transferred after detachrnent by 10 mM tris(hydroxymethyl)aminomethane-hydro-
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EDTA-trypsin solution containing 0.14 M NaCl, 5.4 chloride, pH 7.4; 1.5 mM MgCl2] containing 0.5% Non-
mM KCl, 5.5 mM glucose, 0.53 mM EDTA (pH 7.2), idet P-40. The suspension was vigorously pipetted and
and 500 ,ug of trypsin per ml. then centrifuged at 800 x g at 4°C. The resultant
Unlabeled AMD was obtained from Serva (Heidel- nuclear pellet was suspended in high-ionic-strength
berg, West Germany); unlabeled colchicine was ob- buffer [0.5 M NaCl; 50 mM MgCl2; 10 mM
tained from Sigma Chemical Co. (St. Louis, Mo.); tris(hydroxymethyl)aminomethane - hydrochloride,
[3H]AMD (3.3 Ci/mmol), methoxy-[3H]colchicine (5.9 pH 7.4] and digested with 100 Mug of ribonuclease-free
Ci/mmol), [5-3H]uridine (11 Ci/mmol), [6-3H]thymi- deoxyribonuclease at room temperature. The diges-
dine (23 Ci/mmol), [3H]glucosamine (21 Ci/nmol),DL- tion was stopped by adding SDS and EDTA up to
[3H]valine (0.39 Ci/mmol), and [3H]hystidine (40 Ci/ 0.5% and 10 mM, respectively. After reconstitution
mmol) were purchased from the Radiochemical with the cytoplasmic fraction, the RNA was extracted
Centre (Amersham, England); cytochalasin B was ob- by the phenol-chloroform-isoamyl alcohol method
tained from Aldrich Chemical Co. (Milwaukee, Wis.). (30).
Trypsin (3x crystallized) was purchased from Worth- Evaluation of acid-soluble pool of uridine nu-
ington (Freehold, N.J.). cleotides. The acid-soluble pool was extracted from
Determination of uptake, efflux, and intracel- the cells with 0.5 N perchloric acid, and its components
lular AMD concentration. Confluent monolayers in were separated by ascending paper chromatography
Linbro plates were exposed to [3H]AMD, if required, in ethanol-ammonium acetate, essentially as de-
and unlabeled AMD at the concentrations indicated scribed by Plagemann et al. (26).
in the single experiments. At different time intervals, Assay of uridine kinase. The preparation of the
monolayers were carefully washed with phosphate- cell-free extract and the assay of uridine kinase were
buffered saline and dissolved in sodium dodecyl sulfate performed as described by Plagemann et al. (26). The
(SDS) buffer (3), and the radioactivity incorporated protein content of the cell-free extract was determined
into whole cells was estimated. The AMD intracellular by the method of Lowry et al. (23).
concentration was calculated on the basis of previous Labeling of surface components in trypsinate-
assumptions (3). To determine AMD efflux, cells were and EDTA-extracted trichloroacetic acid-precip-
loaded for 1 h with [3H]AMD as described above, and itable materials. Confluent C and A70 monolayers in
then washed with cold MEM containing 5 ,ug of unla- Linbro plates were exposed to either [3H]glucosamine
beled AMD per ml; prewarmed MEM was then added. (20 MuCi/ml) or to a mixture of [3H]lysine and [3H]-
At different times thereafter the cellular radioactivity valine (both 10 MCi/ml) in MEM without or with
was measured. AMD at 0.5 ,ug/ml. At intervals during incubation,
The nucleocytoplasmic distribution of AMD was monolayers were washed four times with phosphate-
measured as reported elsewhere (3). buffered saline and subjected to the following alternate
In some experiments, the influx of AMD was mea- procedures: (i) digestion with 0.5 Mug of trypsin per ml,
sured after treatment of Linbro monolayers with either 5 min at 37°C, the incubation being stopped by addi-
EDTA (10-2 M) in serum-free MEM or purified tryp- tion of excess soybean inhibitor; samples of this ex-
sin (0.5 mg/ml). tract, called "trypsinate," were then counted for radio-
Uridine incorporation into total cell and acid- activity incorporated; or (ii) detachment with 102 M
precipitable material. Serum-free MEM containing EDTA in serum-free MEM, the reaction being stopped
10lCi of [3H]uridine per ml was added to the confluent at min 5 by addition of a mixture of Ca2" and Mg2"
monolayers in Linbro plates; at different time intervals gluconate, 5 mM (17). This EDTA extract was precip-
the incorporation was stopped by washing with cold itated with 10% vol/vol trichloroacetic acid, collected
phosphate-buffered saline, and monolayers were on Millipore filters, and counted for radioactivity in-
dissolved with SDS-EDTA-papain buffer [10 mM corporated.
NaCl; 10 mM tris(hydroxymethyl)aminomethane-hy- The cellular pellets recovered from procedures (i)
drochloride, pH 7.0; 1 mM MgCl2; 10 mM EDTA; 0.5% and (ii) were subjected to the following two treat-
SDS; 20 Mug of papain per ml; and 20 ,ug of cysteine per ments: (iii) glucosamine-labeled cells were dissolved in
ml]. SDS-EDTA buffer and precipitated on Millipore fil-
Samples of this lysate were precipitated on 0.45-,um ters, as described above; and (iv) amino acid-labeled
membrane filters (Millipore Corp., Bedford, Mass.) cells were lysed with 1.0 N NaOH plus 1% (wt/vol)
with trichloroacetic acid at a final concentration of SDS in water and trichloroacetic acid precipiated for
10%. The filters, washed with 5% trichloroacetic acid radioactivity determinations, as described above.302 BENEDETTO, CASSONE, AND DELFINI ANTIMICROB. AGENTS CHEMOTHER.
Colchicine binding assay and tubulin purifi-
cation. Standard tubulin was purified from pig brain
according to Weisenberg et al. (31) and stored at
-80°C in M sucrose solution according to Lee et al.
(22).
The colchicine binding assay on both pig brain
tubulin and 110,000 x g supernatant fraction from 37
RC cells (control and AMD treated) was performed
according to Weisenberg et al. (31).
Electron microscopy. Cells on Falcon plastic sub-
strata were prefixed with a mixture of 2.5% glutaral-
dehyde (vol/vol) and 1% (vol/vol) TAPO (tris-1-azir-
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idinyl phosphine oxide, Polysciences, Warrington, Pa.)
in 0.1 M phosphate buffer (pH 7.2). After buffer wash-
ing, they were postfixed with 1% (wt/vol) 0804 in the
same buffer for 6 h at 4°C, then processed for electron
microscopy as described elsewhere (13). Sectioning
with a Porter-Blum MT2 ultramicrotome equipped
with a diamond knife was carried out parallel to the
plastic substratum, and then sections were stained
with lead citrate and uranyl acetate (13) and observed
under the Siemens LA electron microscope operating a)
at 80/100 kV. _
Radioactivity determination. Radioactivity was
determined in Bray solution with a Beckman liquid CU
scintillation system MD LS-133. Counting efficiency 0
was calculated by the two-channel ratio using the
external standard method.
RESULTS
Cellular viability and macromolecular Time (days)
synthesis after AMD treatments. 37 RC cells FIG. 1. Effect of AMD on growth curve and viabil-
treated with 0.1 to 0.5 ,ug of AMD per ml (doses ity of 37 RC cells. Cells were seeded in Linbro plates
which in 30 min inhibit the RNA synthesis by at the indicated concentration and detached daily by
60 to about 80%), called A cells, were assayed trypsin-EDTA treatment. The viability was esti-
every 12 h over a period of at least 48 h for mated by suspending cells in MEM containing I mg
cellular viability and deoxyribonucleic acid of trypan blue per ml at 37°C for 15 min. Cells were
(DNA) and RNA synthesis as compared to un- then counted in a hemochromocytometer slide cham-
treated cells, called C cells. At these concentra- ber. The arrows indicate the time of AMD addition
tions the degree of killing by AMD was found to (0.5 pg/ml) to some monolayers (0); control mono-
depend more on the growth stage at the begin- layers (0) received MEM only. The inset shows the
time course of [3H]thymidine (0.5 uCi/ml) incorpo-
ning of the treatment than on the length of the ration into acid-precipitable material (see uridine
treatment itself. A marked fall in the number of incorporation in the text) starting at h 48 from seed-
viable cells occurred when the treatment began ing in control (0) and AMD-treated (0) cells.
at the middle exponential phase of growth (Fig.
1), whereas viability was largely maintained if
cells were treated during their stationary phase sensitive to AMD. This increase in RNA synthe-
of growth. In this latter case the constancy of sis was maximal at h 70 (Table 1).
the number of viable cells did not reflect a This proved, however, to be a merely quanti-
steady-state equilibrium between cell multipli- tative phenomenon, since it could be overcome
cation and AMD-induced lethality, since the by increasing AMD concentration in the me-
cells, after a lag of 6 to 8 h, were no longer able dium; at 20 ,ug of AMD per ml the RNA labeling
to enter the S phase, as demonstrated by the of cells treated for 70 h (A70 cells) was again
low, basal level of [3H]thymidine incorporation completely suppressed (Fig. 2).
into DNA (Fig. 1, inset). The examination of the acid-soluble uridine
The monitoring of [3H]uridine incorporation pool and of kinase activity showed that A70 cells
into cellular RNA during AMD treatment took up uridine efficiently, though at a lower
showed that RNA synthesis continued to be initial rate than C cells. Furthermore, they did
strongly affected until about h 45 to 50, when not differ significantly from control cells in the
labeling was unexpectedly found to increase ability to phosphorylate the precursor and in the
markedly as if synthesis had become much less distribution of the label among the various com-VOL. 15, 1979 AMD RESISTANCE OF 37 RC CELLS 303
ponents of uridine nucleotide pool (data not From these data it seems that the phenome-
shown). The same holds true for cells at inter- non was a phenotypic one dealing with the whole
mediate times of AMD treatment. The increase or the largest part of the population and not
of RNA labeling in A70 cells could not be ac- involving the selection of preexisting AMD-re-
counted for by an increase in the transport or sistant mutants (16) (see below for additional
phosphorylation of the labeled precursor. evidence).
If A70 cells were left in AMD-free medium, Intracellular AMD concentration versus
they recovered, in a period of 36 to 48 h, the full RNA synthesis in A cells. The differences in
capacity of RNA synthesis; this was again as the AMD sensitivity of transcription in C and
sensitive to the drug as usual (Table 1). A70 cells might be related to variations in the
intracellular concentration of the drug. First of
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80 all, we established that AMD was not degraded
z or inactivated by A cells (as seen by incubation
of AMD with extract of A cells and subsequent
paper chromatography). The alternative hy-
60 pothesis was that the steady-state equilibrium
Q. between influx, binding, and efflux of AMD
might alter so that the level of intracellular
AMD diminished. That this was indeed the case
40 is shown in Fig. 3. In fact, given an external dose
of 0.5 ,ug of AMD per ml, the intracellular drug
20 concentration reached its equilibrium at about
90 min, with a value of 0.026 nmol per 106 cells.
.*' 20_ Starting from about h 24, this value decreased,
dropping to about 0.010 nmol per 106 cells at h
70 (Fig. 3). That this fall is sufficient to account
for the late resurgence of transcription is shown
C
4 8 f2 16
AMID concentration in the medium (,pM)
0,, 3
FIG. 2. Dose-response curve of [3H]uridine incor-
poration into RNA in C and A70 cells versus AMD. C
(0) and A70 (0) cells were pulsed with [3H]uridine
(5 uCi/ml) for 30 min in the presence of various doses
of AMD; then the RNA was extracted, and the asso-
ciated radioactivity was measured as described in
the text.
TABLE 1. Monitoring of RNA labeling during AMD
treatment and recovery
E0 I\
[;'H]uridine .h.t.
Cells' (cpm) incorpo- Inhibtion C=
rated into RNA8 )
C (untreated) 55,800 _ 2
Ao.5 9,000 83.8
A24 4,500 91.9
A48 12,300 78.0
A70 33,480 40.0
Aree 48 52,300 6.3
Ares 48 plus AMD 8,000 85.7
(0.5 [Lg/ml)
aCells were treated with 0.5 ytg of AMD per ml and 0.5 10 1.5 24 48 72
called A cells; the subscript number denotes the time
(in hours) of treatment. Ar,, denotes cells treated with Time of treatment (hours)
AMD for 70 h, washed free of the drug, and left to FIG. 3. Monitoring of intracellular AMD amount
recover for the indicated time. during long AMD treatment of 37 RC cells. 37 RC
b The labeled precursor, at a concentration of 10 cultures in Linbro plates were exposed to 1,32 uCi
,ICi/ml, was given to the cells for 30 min. Results are (corresponding to 0.5 pg/ml) of [3H]AMD per ml. At
the mean of three independent experiments. For any the indicated time the intracellular AMD was mea-
other details, see the text. sured as described in the text.304 BENEDETTO, CASSONE, AND DELFINI ANTIMICROB. AGENTS CHEMOTHER.
in Fig. 4, where the percentage of RNA synthesis
is monitored against both the extracellular and
the correspondent proportional intracellular
AMD concentration. It is seen that AMD inhi-
bition of transcription quickly drops as the in-
tracellular drug concentration falls below 0.02
nmol per 106 cells (Fig. 4).
Uptake, influx, and efflux of AMD.To get
insight into the mechanism of the observed dim-
inution in the intracellular concentration of
AMD during AMD treatment of 37 RC cells, we
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compared the uptake and the efflux of the drug
in C and A70 cells. Am0 cells took up in 1 h half as
much AMD as did C cells (Fig. 5), whereas they
released the drug at a comparable rate. Cells
treated with AMD for 2 h (A2 cells) and having
a higher endocellular amount of drug than A70
cells (see Fig. 3) took up and eluted the drug at
a rate similar to that of C cells, showing that the
presence of intracellular AMD does not influ-
ence per se the uptake of other drug.
It is also evident from Fig. 5 that the early
FIG. 5. Time course of AMD uptake and efflux in
C, A2, and A70 cells. 37 RC cultures in Linbro plates
O0 2.0 were exposed to 5 ,uCi of [3HJAMD per ml plus un-
labeled AMD up to a final concentration of 5 pg/ml.
Part of the monolayers (C cells, 0; A2 cells, A; A.o
-I;
cells, *) were measured for AMD uptake at the indi-
8 1. 6 E cated times. Other monolayers preloaded for 60 min
0oJ t- with AMD were washed free of the drug and left to
0
A
c release AMD (C, A2, and A70 cells; same symbols as
above). At the indicated time intervals, samples were
12 > measured for AMD intracellular concentration as
E 6 c
described in the text.
4a
0
noa
'0 4 0 * 0.8 :::
uptake is much lower in A70 cells, and this was
c
C)
V- C
ca. confirmed by a closer examination of the initial
0 L- uptake rate of AMD. We assumed (like others)
21 0@ 0.4 . (8) as "initial" the amount taken up at 2 min,
and called it "influx" rate even though one can-
not be certain, on the basis of the kinetics evi-
dence only, that even at very early times, the
20 40 60 rate-limiting step is the permeation and not the
Percentdge of control RNA synthesis binding of the drug.
FIG. 4. The relationship between external and in-
The influx rate of AMD, both in C and A cells,
tracellular concentration of AMD at equilibrium and was proportional to the external AMD concen-
inhibition of RNA synthesis. Some confluent mono- tration up to 6 uM (Fig. 6). This suggests a
layers of 37 RC cells in Linbro wells were treated for mechanism of passive diffusion for drug entry in
90 min with 1 ,uCi of [3H]AMD per ml plus various 37 RC cells and seems to exclude the existence
amounts of unlabeled drug up to indicated concen- of any high-affinity carrier.On the other hand,
trations (right ordinate); other monolayers were ex- the influx of AMD was seen to decrease progres-
posed to unlabeled AMD only, at identical concentra- sively during the treatment of 37 RC cells with
tions (see above) for 80 min, then pulsed for 10 min the drug (0.5 ,g/ml). The decrease apparently
with 10,uCi of [3Hluridine per ml. The first monolayer started at about h 10 and was maximal at h 70,
was measured for AMD intracellular concentration
(left ordinate), and the second set was processed for when approximately threefold reduction in the
uridine incorporated into trichloroacetic acid-precip- influx was observed. If the nucleocytoplasmic
itable material. The obtained values were expressed distribution of AMD was measured at 2 min, it
as percentage of RNA synthesis in untreated cells. was seen that, over a range of AMD concentra-
For any other details, see the text. tion, the AMD amount in the cytoplasm of bothVOL. 15, 1979 AMD RESISTANCE OF 37 RC CELLS 305
C and A cells was about six times that in the cells, exactly parallels the decrease of incorpo-
nucleus (Fig. 7). Since in 37 RC cells the amount ration into the respective cytoplasm.
of AMD at equilibrium is higher in the nucleus The progressive decrease of the influx rate of
(60%) than in the cytoplasm (40%) (3), it seems AMD in A cells could be mediated by the par-
justified to consider the uptake rate at 2 min as allel decay of a hypothetical membrane carrier
largely representative of the net influx rate. In responsible for AMD entry in these cells. It
this line, it should also be noted that the de- should be recalled here that, even though the
crease in the rate of AMD incorporated into the curves in Fig. 5 are consistent with a simple
nucleus of A70 cells, as compared to that of C diffusion mechanism of entry, the existence of a
very low-affinity carrier for the drug cannot be
excluded on the basis of kinetics evidence only
(8).
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If AMD were acting by decay of a membrane
carrier, it should be expected that inhibition of
protein synthesis mimicked the effect. Against
this hypothesis, it was seen that the influx rate
of AMD was not influenced by cycloheximide
used for 28 h at a concentration sufficient to
block more than 98% of protein synthesis (Table
2).
Taken together with the kinetics of AMD
entry, these data made improbable the hypoth-
esis that the decay of AMD influx is caused by
the loss of a specific carrier protein having very
low affinity for the drug.
Morphology of 37 RC cells after pro-
longed AMD treatment. To get general insight
into the mechanism of the reduction of AMD
AMD concentration in the mediurn (, M) uptake into 37 RC cells, some cultural and mor-
FIG. 6. Initial rate of AMD uptake in C and A phofunctional parameters, as well as the ultra-
cells. 37 RC cultures in Linbro plates (C, 0; A12, 0; structure of cells after prolonged AMD treat-
A,a, A; A70, A) were exposed to MEM containing ment, were considered.
[3HJAMD concentrations ranging from 1.5 to 6 W. Relevant changes in these parameters were
After 2 min, the amount of intracellular AMD was indeed observed, many of them seemingly re-
measured as described in the text. The initial rate is lated to the cell surface. By phase-contrast mi-
expressed as millimoles x 10-8 of AMD taken up per croscopy, A70 cells appeared more flattened and
106 cells in 1 min.
highly contrasted at their margins. Their adhe-
siveness to glass or plastic surfaces was clearly
increased, as demonstrated by the longer time
Nucleus
required for their detachment by trypsin-EDTA
solution. Once detached, their rounding was de-
layed, and they maintained an elongated form
longer than C cells. Finally, blebs or villus-like
protrusions were not so numerous in A as in C
cells, as if the former had a more rigid cell
periphery (see Fig. 8).
TABLE 2. Effect of treatment with various
inhibitors on the influx rate of AMD in 37 RC cells
AMD incorporated in
Treatment 100 s (mmol x 10'
15 225 30 075 15 225 30
per 10'i cells)'
AMD Concentration in the medium (,uM)
None ........................ 4.1 ± 0.50
FIG. 7. 37 RC in Linbro plates (C, 0; A70, 0) were AMD (0.5 ,ug/ml for 24 h) ...... 2.8 ± 0.32
exposed to MEM containing [3H]AMD concentra- Cycloheximide (25 ,og/ml
tions ranging from 0.75 to 3 WM. After 2 min, incor- for 28 h) .... 4.3 ± 0.41
poration was stopped by washing with cold MEM, Colchicine (1 isg/ml for 12 hr) 4.6 ± 0.52
cells were detached by EDTA-trypsin, and the radio-
activity incorporated into the nucleus and into the 0The values are the mean of three independent
cytoplasm was measured as reported in the text. experiments, expressed as ± standard deviation.Downloaded from http://aac.asm.org/ on March 17, 2021 by guest
FIG. 8 a-c
306
,
J1
4'VOL. 15, 1979 AMD RESISTANCE OF 37 RC CELLS 307
'SA ''
-~~
~~4~~' -'~1
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FIG. 8 d
Under the electron microscope, the nucleus of richer in intracytoplasmic membranous ele-
A70 cells showed the distinct effects of AMD, ments. In particular, several stacks of parallel
already well established (5, 18) due to the block membranes of the endoplasmic reticulum sys-
of nucleolus physiology, confirming that ribo- tem were seen in the perinuclear region, whereas
somal RNA synthesis was inhibited under our C cells showed only very few scattered rough
conditions. Major changes, however, were seen and smooth endoplasmic reticulum profiles (Fig.
in the cytoskeleton components of A cells. Mi- 8d). In some perinuclear areas of A cells, mem-
crotubules were apparently greatly diminished, brane elements resembling annulate lamellae
whereas microfilaments (4.0 to 6.0 nm) were were also observed (Fig. 8e). Another distinctive
markedly more numerous and were mostly seen aspect of A70 cells was their higher content of
in the form of prominent bundles just beneath Golgi bodies, more often seen in the perinuclear
the plasma membrane (Fig. 8b). Filamentous region (Fig. 8e).
structures were more concentrated at the dish- Effect of cytochalasin B, EDTA, and tryp-
adhesive side of the cells, and the contact surface sin on influx rate of AMD in C and A cells.
in flat-embedded sections appeared rich in coat The results reported in the preceding sections
components (Fig. 8c). prompted us to study the possible influence of
The scanty appearance of microtubules in surface-modifying agents and cytoskeleton dis-
AMD-treated cells was paralleled by diminution organizers on the entry of AMD in 37 RC cells.
of colchicine-binding proteins (tubulin) in the To this end, cells were brought into suspension
supernatant fraction of these cells (Table 3). from monolayers using different concentrations
While being poor of ribosomes, A70 cells were and times of treatment of both EDTA and tryp-
FIG. 8. Electron micrographs of C (a) and A70 cells (b - e). (a) and (b) show adjacent cells. Conspicuous
bundles of 6.5 to 4.0-nm- thick microfilaments (arrows) are seen in A70 cells, just beneath the plasma membrane
and parallel to it (b). Note also the scattered free ribosomes and polysomal complexes in cells (a. thin arrows)
and their virtual absence in the cytoplasmic areas of A70 cells (b). Complexes of parallel membranes (CM) as
well as annulate lamellae (AL) are seen in perinuclear areas of some A70 cells (d and e). Golgi bodies (GB) are
also more numerous in A70 cells (e) than in C cells (c) A70 cell in a flat-embedded section, showing the
accumulation of filaments (arrows) in the cytoplasm oriented toward the plastic surface (bottom). Other
abbreviations: N, nucleus; Nu, nucleolus; RER, rough endoplasmic reticulum; M, mitochondria. Bar, 100 nm.308 BENEDETTO, CASSONE, AND DELFINI ANTIMICROB. AGENTS CHEMOTHER.
-Al~
~~ ~ ~ ~
m~~~~a
iZSa" ''
~~~~~~~~~~~~~~~~~~~w~~~~~~
t
3r~~~~~~~~~~~;
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S,SK :~t,'
S"7 2K>I.
*44
*A`
r~~~~A
IN,~~~~~~~~~~~~~~~~~~~~9y
la
(i
A4 I1..o
ti
44''~~~~~~~~~~~~~~4
0
FIG. 8 e
sin, two classical cell-detaching agents which act solutions, under conditions of maintenance of
by removal of the surface coat and membrane full viability, no significant modification of AMD
materials. influx was observed. On the contrary, similar
After complete detachment and suspension of treatments performed with A70 cells caused a
control cells by both EDTA and EDTA-trypsin marked increase in rate of the AMD influx asVOL. 15,1979 AMD RESISTANCE OF 37 RC CELLS 309
cells were detached from the dish. Finally, after by Johnson and Griffin (17), some of the mate-
EDTA-trypsin treatment, the influx of the drug rial contained in these preparations could be
into A70 cells reached values comparable with constituted of integral membrane proteins. The
those of suspended control cells (Table 4). radioactivity incorporated as both glucosamine
After suspension, therefore, A cells behaved and amino acids in "stripped-off' A70 cells was
like C cells in AMD entry. This was confirmed about two times less than in controls (Fig. 9). On
at various external AMD concentrations with the other hand, the radioactivity incorporated
dose-response curves, like those shown in Fig. 5, into surface proteins was practically the same in
being identical for C and A cells. both cells, regardless of the way by which they
On the other hawnd, the use of cytochalasin B, were freed from these proteins (trypsin or
an inhibitor of microfilament organization, did EDTA). This means that, although total protein
not cause either In C or in A cells any significant synthesis decreased as expected in AMD-treated
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modification of the the influx rate of AMD cells, in these latter a greater portion of the
(Table 4). The lack of effect of cytochalasin B residual synthesized protein is glycosylated and
was the same in monolayers as in suspended exported to the cell surface. Indeed, the ratio
cells, a fact especially relevant because it is between external and internal proteins is defi-
known that the simple detachment from the nitely higher in A70 cells (Fig. 9).
dish of epithelial cells like BHK-21 and BSC 1
provokes the disarrengement of microfilament
bundles (15).
Finally, treatment of 37 RC cells with colchi-
cine, a specific inhibitor of microtubule poly-
merization, did not affect the influx rate of AMD
over the time interval of 12 h (Table 2).
Labeling of surface components in C and
A70 cells. On the basis of previous results it was
of interest to look at possible differences in the
rate of synthesis and secretion of surface com-
ponents in C andA70 cells. To this end we com-
pared the rates of incorporation of radiolabeled
glucosamine and amino acids into total cell and
surface protein. As "surface proteins" we consid-
ered all radioactive materials detached by
EDTA and trypsin solutions, even though they
certainly contained nonprotein components (for
instance, glycolipids and others), As emphasized
TABLE 3. Colchicne binding activity in 37 RC cells
after differernt times of treatment with AMD (0.5
pg/ml) Time (hours)
methoxy,[3H]colchicine bound to FIG. 9. Glycoprotein synthesis and export to cell
Time of treatment DEAE-c,llulose Chromedia filters surface. C and A70 cells in Linbro plates were exposed
(opm/mg of protein)' to MEM containing [H]glucosamine (a and b) and
C cells ... 16,000 CHlvaline plus hystidine (c and d). At different time
A48 11,000 intervals, the radioactivity incorporated into trypsi-
..........
nate (0), trichloroacetic acid-precipitable EDTA-ex-
A70.o .. .. 6,500 tract (A), and residual (O) cell materials was mea-
q DE:AE, Diethylamipoothyl. sured as described in the text.
TABLE 4. Effect of detach/ing treatments with EDTA or trypsin solutions on influx rate of AMD in C and A
cells, pretreated or untreated with cytochalasin Ba
C A70
Cells Detaching agent
-cyt B +cyt B -cyt B +cyt B
Cells in monolayer 5.8 5.0 2.1 1.9
Cells in suspension EDTA 5.9 5.3 4.2 4.1
Trypein 5.1 4.9 5.3 5.3
l.g
aCells were treated for 60 min with 10 of cytochalasin B (cyt B) per ml, respectively, in monolayer or
suspension conditions. Data are expressed as millimoles x 10-8 per 10' cells, incorporated in 100 s.310 BENEDETTO, CASSONE, AND DELFINI ANTIMICROB. AGENTS CHEMOTHER.
DISCUSSION longed treatment with AMD, 37 RC cells are
In this research we have observed that, during able to form a permeability barrier against the
long exposure to AMD, 37 RC cells progressively drug and that this barrier is removable by
acquired the ability to reduce the uptake of the EDTA-trypsin. This does not necessarily imply
drug and so, at a later stage, resume net RNA that the barrier is in the membrane, because it
synthesis. Thus, they recovered transcription could also be represented by cytoplasmic struc-
ability without drug removal, a situation that tures which could control the membrane func-
seems unique for this type of cell. tions.
Two main factors have been seen to be critical Nevertheless, a hypothesis that logically fol-
for this phenomenon to occur: (i) the amount of lows from the discussion above is that the barrier
AMD given to the cells as related to the resulting is constituted of surface glycoprotein materials.
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inhibition of RNA synthesis; and (ii) the state of It is well known that both EDTA and trypsin
the cells at the beginning of the treatment. free such materials from the surface (9), and we
Therefore, doses of AMD must be used which have shown that in A70 cells a greater proportion
do not cause inhibition of transcription greater of total cell proteins is exported to the cell sur-
than 60 to 80%. Moreover, cells must be treated face as glycoproteins, in spite of the lower rate
with AMD when they are confluent, since prior of protein synthesis. A clear ultrastructural
exposure at the stage of exponential growth counterpart to this finding comes from the ob-
caused a significant killing even at the lowest servation that A70 cells are significantly enriched
AMD doses (0.1 ,ug/ml). The lower AMD toxic- in endoplasmic reticulum membranes and have
ity for confluent, topoinhibited 37 RC cells does also more numerous and prominent Golgi bod-
not appear to depend on G. state per se. In fact, ies, which are known as cell organelles, devoted
other cell lines, e.g. chick embryo, human W138, to the glycosylation of the proteins to be secreted
and mouse 3T3 fibroblasts, which can be ar- and organized at the cell surface.
rested in G. state, are highly AMD-sensitive at In keeping with the generality of these obser-
confluence (3), whereas cells that have a high vations, an overproduction, but, possibly, also a
mitotic index and are incapable of reaching a different distribution or a qualitatively different
persistent G. state, like mouse L strains (27) and composition of cell surface coat component
various transformed cell lines, are somewhat less would create the obstacle to AMD influx in A
sensitive to AMD (11, 34). cells. It should be stressed that an accumulation
It is more difficult to find an explanation for of glycoproteins at the cell surface could also be
the reduced AMD uptake by the cells. The due to a selective inhibition of glycosidases, a
uptake, in fact, is the sum of both the transport hypothesis which warrants further investigation.
and the binding and, owing to the great number The findings above also help to give a reason
and the marked avidity of intracellular AMD- for the increased adhesiveness and flattening in
binding sites, it is almost impossible that purely culture of A70 cells, since these seem to be me-
kinetics measurements distinguish between diated by glycoproteins both excreted from and
them. bound to membranes (12, 31).
Neverthless, both the experiments on the nu- In this connection, interesting analogies can
cleocytoplasmic distribution and AMD after 2 be made between 37 RC cells and those special
min (as opposed to the distribution at equilib- cell lines, which, by cultivation in the presence
rium) and the experiments on AMD uptake after of high amounts of AMD, have been selected
EDTA and trypsin strongly suggest that a re- from mutants refractory to the drug (6, 16, 20).
duction in the permeation of the drug is the These cells, indeed, had practically lost the
cause of the progressive lowering of the uptake permeability to the drug; Bosmann has recorded
in AMD-treated cells. several biochemical changes in the plasma mem-
The only other possible alternative is that A70 brane of these cells, including an increase of
cells have less, or less avid, intracellular AMD- glycoprotein synthesis and its deposition at the
binding material. For instance, this could be due cell surface (7). In AMD-resistant hamster ovary
to a decreased cellular volume or a diminution cell lines, a quantitative relationship between
in the amount of the membrane or other un- the presence of an integral membrane protein,
known intracellular binding sites for AMD. molecular weight 170,000, and permeability to
However, we didn't find evidence for any of AMD and other amphophilic drugs has been
these events. In particular, A70 cells were seen to postulated (19). Preliminary experiments of
be a bit larger than C cells, and their intracellular plasma membrane protein characterization in 37
membranes were collectively augmented (see RC showed the presence of a 165,000 molecular-
below). weight protein, but we didn't find any significant
Thus we suggest as a more probable explana- change in the amount of this specific protein
tion of the results obtained that, during a pro- during AMD treatment.VOL. 15, 1979 AMD RESISTANCE OF 37 RC CELLS 311
Another clue for the explanation of the re- in 37 RC cells after actinomycin D inhibition. J. Cell
Biol. 67:538-550.
ported findings arose from the electron micro- 3. Benedetto, A., C. Delfini, S. Puledda, and A. Sebas-
scopic observation that AMD-treated cells were tiani. 1972. Actinomycin D binding to 37 RC and HeLa
also enriched in bundles of microfilaments, likely cell lines. Biochim. Biophys. Acta 287:330-339.
to be actin in nature and mostly running beneath 4. Benedetto, A., and W. Djaczenko. 1972. Cells rapidly
recover their RNA synthesis after inhibiting with high
the plasma membrane. Microfilaments, indeed, doses of actinomycin D. J.Cell Biol. 52:171-174.
have been increasingly associated with the con- 5. Bernhard, W., and N. Granboulan. 1968. Nucleus (Ul-
trol of several plasma membrane functions, in- trastructure in biological systems), p. 81-149. In A. J.
cluding permeation (33). In epithelial cells lines, Dalton and F. Haguenau (ed.), Electron microscopy of
the nucleolus in vertebrate cells, vol. 3. Academic Press
microfilament bundles have been observed to be Inc., New York.
disarranged during cell rounding and detach- 6. Biedler, J. L, and H. Riehm. 1970. Cellular resistance
ment by trypsin (15). It is also known that actin- to actinomycin D in Chinese hamster cells in vitro:
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like filaments of endothelial and smooth-muscle cross-resistance, radioautographic, and cytogenetic
cells are lost on EDTA treatment (10, 21). studies. Cancer Res. 30:1174-1184.
7. Bosmann, H. B. 1971. Mechanism of cellular drug resist-
The results of experiments using the drug ance. Nature (London) 233:566-569.
cytochalasin B, an inhibitor and disrupting agent 8. Bowen, D., and I. D. Goldman. 1975. The relationship
of microfilament assembly (33), do not lend sup- among transport, intracellular binding, and inhibition
port to a major role of these structures in the of RNA synthesis by actinomycin D in Ehrlich ascites
tumor cells in vitro. Cancer Res. 35:3045-3060.
decreased influx of AMD. In fact, under various 9. Buck, C. A., M. C. Glick, and L. Warren. 1970. A
conditions of cytochalasin B treatment, this drug comparative study of glycoproteins from the surface of
did not modify the entry of AMD either in C or control and Rous sarcoma virus transformed hamster
in A cells. cells. Biochemistry 9:4567-4576.
10. Cooke, P. H., and F. S. Fay. 1972. Correlation between
It should be stressed, however, that these fiber length, ultrastructure, and the length-tension re-
results do not constitute conclusive evidence lationship of mammalian smooth muscle. J. Cell Biol.
against the participation of microfilaments in 52:105-116.
the observed phenomenon since, as pointed out 11. Cremisi, C., G. E. Sonenshein, and P. Tournier. 1974.
Studies on the mechanism of actinomycin D resistance
by Goldman and Knipe, the effect of cytochal- of an SV 40-transformed hamster cell line. Exp. Cell
asin B is "multifacet" and could not directly Res. 89:89-94.
relate to microfilament function(15). Nor can it 12. Culp, L. A. 1974. Substrate-attached glycoproteins me-
be excluded that a structural relationship exists diating adhesion of normal and virus-transformed
mouse fibroblasts. J. Cell Biol. 63:71-83.
between surface glycoprotein and inner microfil- 13. Djaczenko, W., and A. Cassone. 1972. Visualization of
aments, so that both could exert a role in creat- new ultrastructural components in the cell wall of Can-
ing a barrier to AMD entry into cells. dida albicans with fixatives containing TAPO. J. Cell
In conclusion, we have shown that 37 RC cells Biol. 52:186-190.
14. Goldman, I. D. 1973. E. Mihich (ed.), Drug resistance
have the remarkable property of responding to and selectivity, p. 299-358. Academic Press, New York.
AMD with changes that progressively hinder 15. Goldman, R. D., and D. M. Knipe. 1972. Functions of
the uptake of the drug, and we have also sug- cytoplasmic fibers in non-muscle cell motility. Cold
gested possible mechanisms for this. Although Spring Harbor Symp. Quant. Biol. 37:523-534.
16. Goldstein, M. N., K. Hamm, and E. Amrod. 1966.
we have detected this property only under a Incorporation of tritiated actinomycin D into drug-sen-
well-defined set of experimental conditions, we sitive and drug-resistant HeLa cells. Science 151:
believe it to be of general relevance for the 1555-1556.
mechanism of AMD resistance of AGMK cell 17. Johnson, H. V., and J. M. Griffin. 1977. Isolation char-
acteristics of HeLa surface proteins. Exp. Cell Res. 109:
lines. In fact, the progressive lowering of the 223-236.
intracellular amount of the drug may contribute 18. Journey, L. J., and M. N. Goldstein. 1961. Electron
to the lower cytotoxicity and to the recovery of microscope studies on HeLa cell lines sensitive and
RNA synthesis shown by these cells after drug resistant to actinomycin D. Cancer Res. 21:929-932.
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ACKNOWLEDGMENTS 20. Kessel, D., and H. B. Bosmann. 1970. On the charac-
We gratefully acknowledge the technical assistance of L. teristics of actinomycin D resistance in L 5178 Y Cells.
Gismondi and G. Oberholtzer in this work. Cancer Res. 30:2695-2701.
A. Benedetto is the recipient of supporting grant no. 21. Lauweryns, J. M., J. Baert, and W. De Loecker. 1976.
73.01395.44 from the Consiglio Nazionale delle Ricerche, Italy. Fine filaments in lymphatic endothelial cells. J. Cell
Biol. 68:163-167.
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