[CANCER RESEARCH 41, 2745-2750, July 1981]0008-5472/81 /0041-OOOOS02.00
Comparative Effects of Adriamycin and N-Trifluoroacetyladriamycin-14-
valerate on Cell Kinetics, Chromosomal Damage, and MacromolecularSynthesis in Wfro1
Awtar Krishan,2 Kamla Dutt, Mervyn Israel, and Ram Ganapathi
Comprehensive Cancer Center for the State of Florida, University of Miami Medical School, Miami, Florida 33 1OÕ[A. K., f>. G.]. and Sidney Farber Cancer Institute.Boston, Massachusetts 02115 ¡M.l., K. D.]
ABSTRACT
A/-Trifluoroacetyladriamycin-14-valerate differs from Adria
mycin in its rapid intracellular transport and lack of fluorescentbinding to nuclei or chromosomes. Both of these anthracyclinescause inhibition in the incorporation of labeled precursors intonucleic acids, extensive chromosomal damage, and arrest ofcell cycle traverse in G2. In human lymphoid cells, /V-trifluo-roacetyladriamycin-14-valerate, unlike Adriamycin, does notshow cell cycle phase-specific or proliferation-related cytotoxiceffects. In an L1210 soft-agar assay, both Adriamycin and N-trifluoroacetyladriamycin-14-valerate show no enhanced sensitivity of mid-S-phase cells to their cytotoxic action.
INTRODUCTIONADR,3 a widely used anthracycline antitumor antibiotic,
causes inhibition of cellular proliferation in vivo and in vitro (3,4, 25). ADR has been shown to inhibit nucleic acid synthesis(12, 20, 24), to cause chromosomal breaks (28, 29), and toproduce an irreversible block of cell cycle traverse in G2 (15,26).
AD 32 is a recently developed analog of ADR, now in clinicaltrial. This analog is therapeutically superior to ADR and is lesstoxic than is the parent antibiotic in a variety of animal testsystems (5, 8, 21, 27). Pharmacological studies in mice, rats,monkeys, and humans have shown that AD 32 undergoesextensive biotransformation, primarily to N-trifluoroacetyladria-mycin and W-trifluoroacetyladriamycinol. Significant levels of
ADR, however, are not seen in serum, bile, or urine followingadministration of AD 32 (6-11). AD 32 differs from ADR in
some of its biological properties. For example, AD 32 bindspoorly, if at all, to isolated calf thymus and other DNA preparations (22); AD 32 is rapidly transported into cells, whereasintracellular transport of ADR is slow and temperature dependent (20). Cells incubated with ADR show gradual appearanceof nuclear and cytoplasmic fluorescence. In fixed cells stainedwith ADR, chromosomes and nuclei are brightly fluorescent. Incontrast, cells incubated with AD 32 show rapid appearance ofcytoplasmic fluorescence and, even after prolonged incubation, no fluorescence can be detected in nuclei or chromosomes of live or fixed cells (17).
' This investigation was supported by USPHS Grants CA19118, CA23688,
and CA29360.2 To whom requests for reprints should be addressed, at Comprehensive
Cancer Center for the State of Florida, University of Miami School of Medicine(R-71), P. O. Box 16960, Miami, Fla. 33101.
3 The abbreviations used are: ADR, Adriamycin; AD 32, W-trifluoroacetyladria-mycin-14-valerate; ID50,50% inhibitory dose; IMPY, 2,3-dihydro-1 H-imidazo[1.2-bjpyrazole.
Received October 12, 1978; accepted April 15, 1981.
The present investigation was undertaken to compare theeffects of ADR and AD 32 on macromolecular synthesis, cellcycle traverse, cell cycle phase-specific cytotoxicity, and chro
mosomal damage of lymphoid cells in culture.
MATERIALS AND METHODS
Cell Cultures. Log-phase cultures of human lymphoid cellsof T-cell (CCRF-CEM), and B-cell (LAZ-007) origin (obtained
from Dr. H. Lazarus, Sidney Farber Cancer Center, Boston,Mass.) and L1210 mouse leukemic lymphoblasts were grownin stationary or roller bottle cultures and nourished with Eagle's
minimal essential medium supplemented with 10% fetal calfserum and antibiotics. Plateau-phase CCRF-CEM cells were
obtained by allowing cultures to reach a saturation density of3 to 4 x 106 cells/ml over a period of 3 to 4 days; as reported
before (15), these cultures have reduced incorporation of labeled precursors into DNA (approximately 10% of log-phase
cultures), and flow cytometric analysis shows that more than90% of the cells have G, DNA content.
Synchronized cell populations were collected after centrifugal elutriation of log-phase L1210 cells in a Beckman JE-6elutriator rotor in a J21-C centrifuge (Beckman Instruments,Palo Alto, Calif.). Samples (0.5 to 1.0 x 108 cells in 0.85%
sodium chloride solution and 10% calf serum) were loaded intothe JE-6 rotor separation chamber at a flow rate of 10 ml/min
and a rotor speed of 2000 rpm. Various subpopulations (inaliquots of 200 ml) were collected by reducing the centrifugerotor speed. The cell cycle phase distribution of the collectedfractions was checked by autoradiography and flow cytometry.
Cell counts (after drug treatment) were determined by incubation of cells for 5 to 10 min in 0.4% trypan blue in 0.85%sodium chloride solution. Stained cells were counted as dead,whereas the dye-excluding cells were counted as living. However, it is understood that a "viable cell" in this assay may not
be clonogenic. A soft-agar colony assay was used to monitordrug effects on L1210 cells. For this procedure, log-phase
L1210 cells or synchronized subpopulations (obtained by elutriation) were incubated in Eagle's minimal essential medium
containing ADR or AD 32 (0.01 to 10 jug/ml) for 1 to 2 hr at37°. Following drug treatment, cells were washed twice in
drug-free medium and recovered by centrifugation at 80 x g.Control and treated cells were then plated in 35- x 10-mm Petridishes using Eagle's minimal essential medium containing 20%
fetal calf serum, 10 /IM 2-mercaptoethanol, and 0.3% agar.Petri dishes were incubated for 7 days at 37°in a humid 95%
air-5% CO2 atmosphere. A colony in this assay was defined as
containing more than 25 cells after 7 days of incubation. Inuntreated L1210 cells incubated for 7 days in soft agar, more
JULY 1981 2745
Association for Cancer Research. by guest on September 1, 2020. Copyright 1981 Americanhttps://bloodcancerdiscov.aacrjournals.orgDownloaded from
A. Krishan et al.
than 90% of the colonies had greater than 50 cells. Platingefficiency of these cells varied between 30 and 50% in ourlaboratory.
All drug incubation experiments in stationary cultures andsoft-agar plating assays were set up in triplicate. Cell counts
from the suspension cultures in triplicate did not differ by morethan 5%. In soft-agar assay, colonies were counted under an
inverted microscope at a magnification of x40. Five differentareas (approximately 10 sq mm) selected at random from theplate were counted.
Cell Cycle Analysis. Cell cycle progression was monitoredby laser flow cytometry after staining of samples by the propi-dium iodide-hypotonic citrate method. Details of the staining
method and of the instrumentation have been described (14).Stained samples were analyzed in a Coulter Electronics TPS-Icell sorter interfaced to a Hewlett-Packard 9845A computer.
Software programs developed by Dr. B. Bagwell (2, 23) andCoulter Electronics, Inc., were used for data acquisition, cellcycle analysis, and plotting.
Labeling indices were determined by autoradiography afterincubation of cell aliquots with thymidine (0.1 juCi/ml; specificactivity, 50 Ci/mmol) for 1 hr.
Cytogenetic Analysis. Log-phase cultures of CCRF-CEM
cells were exposed to various drug concentrations for 2 hr,washed, and reincubated in drug-free medium for the next 22
hr. Two hr before termination of the experiment, vinblastinesulfate (Eli Lilly, Indianapolis, Ind.; 0.01 jag/ml) was added tothe cultures for accumulation of cells in mitosis. After hypotonieswelling of the cells in 0.075 M potassium chloride for 30 min,air-dried smears were made and stained with Giemsa stain.
Labeled Precursor Uptake. LAZ-007 cells in log phase (1X 106 cells/ml) were incubated with ADR or AD32 (0.5 to 5.0
jug/ml) for 6, 12, 18, and 24 hr. One hr before removal of cellaliquots, cultures were pulse labeled with tritiated 1 jiiCi/mldoses of thymidine (1.9 Ci/mmol), uridine (8 Ci/mmol), or L-leucine (6 Ci/mmol), all from Schwarz/Mann, Orangeburg, N.Y. Cells were washed by centrifugation and precipitated with5% trichloroacetic acid. The precipitates were collected onmixed-ester filters and processed for liquid scintillation counting (13). Samples were counted in an LS-7000 liquid scintillation counter (Beckman Instruments); cpm/106 cells were
52,180 ± 1,981 (S.D.) for thymidine, 50,000 ± 1,700 foruridine, and 1,782 ±100 for leucine.
RESULTS
Cellular Proliferation. In CCRF-CEM cultures incubated withAD 32 (0.01 ¿ig/ml) for 24 and 48 hr, the number of dye-
excluding cells was similar to that of control cultures. In culturesincubated with AD 32 (0.1 and 1.0 jug/ml) for 24 hr, thenumbers of dye-excluding cells were 60 and 26% of control,respectively. Continued incubation of cells in 1.O-ftg/mlamounts of drug-containing medium resulted in further decrease in the number of dye-excluding cells; after 48 hr, most
of the cells were dead.From these observations and a number of similar and parallel
experiments with ADR, the ID50of AD 32 for log-phase CCRF-
CEM cultures incubated for 24 hr was determined to be between 0.3 and 0.5 /¿g/ml,compared to an ID50 of 0.1 to 0.2jug/ml for ADR under similar conditions.
The viable cell count data for L1210 cells exposed to ADR
and AD 32 were similar to those for CCRF-CEM cells whereas,in the human B-cells (LAZ-007), the numbers of dye-excluding
cells after 24 hr of exposure to 0.1, 1.0, and 5 fig/m\ were 45,23 and 13%, respectively, for ADR and 58, 45 and 10%,respectively, for AD 32. In this cell line, the ID50values for the2 drugs were closer than in the CCRF-CEM cells.
In subsequent experiments, we used L1210 cells and a soft-
agar assay for quantitating drug effects on cellular proliferation.Data in Chart 1 show the effect of ADR and AD 32 on the abilityof L1210 cells to form colonies in soft agar. In this assay, the2- to 3-fold higher cytotoxicity of ADR (as compared to AD 32)seen by the dye exclusion cell-counting methods in L1210 andCCRF-CEM cells and described above was not clearly dem
onstrated. In cells exposed to similar drug concentrations (0.01to 1 /jg/ml), the numbers of colonies counted were approximately 23 to 60% higher in the AD 32-treated cultures than in
those exposed to ADR. In cells incubated with ADR (10 fig/ml), hardly any colonies could be seen (0.3%), whereas a fewcolonies (3%) were still visible after treatment with AD 32.
In our previous study (15), the effects of ADR on cell countsand cell cycle traverse of CCRF-CEM lymphoblasts were shown
to be directly related to drug concentration and length ofexposure. A gradual decrease in cell counts was related to thelength of incubation. For example, in cells exposed to ADR (0.5jug/ml) for 1, 2, or 24 hr, the dye-excluding cell counts were
40, 30, and 20% of control, respectively. Obviously, theseresults were anticipated in view of the slow intracellular transport of ADR. In the present study, similar experiments werecarried out with AD 32 which, in contrast to ADR, is rapidlytransported across the cell membrane.
Data in Table 1 compare the number of dye-excluding cells
(expressed as percentage of control), following exposure ofCCRF-CEM cells to AD 32 concentrations of 0.5, 1.0, and 5.0jug/ml for 15 and 60 min (washed and incubated in drug-free
medium for 24 to 72 hr before analysis). In cultures exposedcontinuously to AD 32 (Table 1, Columns 8 to 10), concentration and exposure time-dependent effects on cell counts were
evident. In cultures exposed to AD 32 (0.5 ng/ml) for 5 min
ONTROL8I.,ufe
«.g«u2
«oo.«rr^—
1•M_J_^1
11ftTiA^^^°
S 2 8 g5288O
O ~ o O <> MO-HIV«fUMZfVIYCZN
ñO-lt
tUQ/»1)
Chart 1. Effect of ADR and AD 32 on the clonogenicity of L1210 cells in thesoft-agar assay. Bars, S.D.
2746 CANCER RESEARCH VOL 41
Association for Cancer Research. by guest on September 1, 2020. Copyright 1981 Americanhttps://bloodcancerdiscov.aacrjournals.orgDownloaded from
ADR and AD 32 in Vitro
Table 1Effect of AD 32 on cell counts of log-phase CCRF-CEM lymphoblasts
No. of dye-excluding cells(%Rein-
cuba-tion(hr)24
487215
minexposure0.5ml79
60401.0/»o/ml8041325.0M9/
ml43
124of
control) at following AD 32doses1
hrexposure0.5f»9/ml81
47551.0ml4718145.0eg/ml3874Continuous
exposure0.5WJ/ml51
1941.0MO/
ml30
815.0
ml23
40
and washed and reincubated in fresh medium, no major effecton cellular proliferation was noted. However, exposure of cellsto higher concentrations (5 |Ug/ml) of AD 32 for 5 min did resultin progressive loss of viability with time, and after 72 hr ofincubation counts were only 9% of control (data not shown inTable 1).
Cultures exposed to AD 32 for 15 min or 1 hr, followed bywashing and resuspension in fresh medium for 24 hr, showedapproximately similar reductions in the number of cells. DNAdistribution histograms of cells exposed to drug for 15 min orfor 1 hr (and analyzed after 24 hr) were also similar.
Metabolic Effects. Data in Chart 2 show that AD 32 andADR concentrations of 1 to 5 jug/ml have a markedly inhibitoryeffect on nucleic acid synthesis. In cultures exposed for 6 hr toADR and AD 32, inhibitory effects on DNA synthesis correlatedwith slowing of the cell cycle traverse and accumulation of cellsin S phase. After 24 hr incubation, most of the cells exposedto ADR (0.1 to 1 fig) or AD 32 (1 to 5 jug) were blocked in G2,with a corresponding reduction in thymidine incorporation.Higher concentrations of ADR (1.0 /¿g/ml)and AD 32 (5 ftg/ml) reduced thymidine incorporation after 6 hr to 6 and 1% ofcontrol values, respectively; the accumulation of cells in G,-early S phase was noted in these cultures. ADR had far moreinhibitory effects on DNA synthesis than did similar concentrations of AD 32.
Both ADR and AD 32 (1 to 5 /¿g/ml) had a significantinhibitory effect on the incorporation of uridine into RNA. Onlyvery high drug concentrations (1.0 /¿gADR and 5 /tg AD 32)had inhibitory effects on the incorporation of leucine into cellular proteins. Maximum inhibition was seen after 6 to 12 hr ofexposure to 5-/¿g/mldoses of ADR and AD 32.
Cytogenetic Effects. As described below, AD 32, by causingan irreversible arrest of cells in G2, prevents cell cycle progression to mitosis. However, occasional mitotic plates wereseen in cultures exposed to AD 32 for short periods of time (2hr) and incubated in the presence of a C-mitotic agent, vin-
blastine. In metaphase plates obtained by this procedure, extensive drug-induced chromosomal damage was observed.
Besides breaks in one or both chromatids, nonstaining gaps,and chromosomal fragments, extensive multiradial translocations involving up to 3 chromosomes were noted.
Table 2 summarizes the chromosomal damage seen in metaphase plates from cultures exposed to ADR (0.01 and 1.0/¿g/ml) and AD 32 (0.5, 1.0, and 10.0 /¿g/ml). Metaphaseplates with chromosomal damage were far more frequent in AD32-treated cultures than in cells exposed to ADR. Similarly, theextent of damage was also greater in AD 32-treated plates thanin ADR-treated plates. However, it is possible that cells with
higher drug-induced damage (e.g., after exposure to ADR) mayfail to reach the mitotic stage and thus not be available foranalysis.
Cytokinetic Effects. AD 32 resembles ADR in causing adrug concentration and exposure time-dependent block of cell
cycle traverse in G2. As shown in Chart 3 (6 and C), cellsexposed to ADR (0.1 to 1.0 /jg/ml for 24 hr) are blocked in G2,whereas a higher ADR concentration [10 fig (Chart 3D)] inhibitscell cycle traverse. A comparison of histograms in Chart 3shows that exposure of cells to AD 32 [0.1 jug/ml (Chart 3F)]slows the cell cycle traverse as indicated by the accumulationof cells in S and exposure to 1.0 /ig/ml (Chart 3G) leads toaccumulation of cells in G2. At a higher concentration [10 jug/
180.
140.
100
60
20
^1
\
12 18 24
O
O
180.
140
100.
60
20U
o.
B
12 18 24
180
MO
100.
60
20 •¿�C
6 12HOURS
18 24
Chart 2. Effect of ADR and AD 32 on the uptake and incorporation of labeledpercursors into DNA (A). RNA (6), and proteins (C) of LAZ-007 cells. The cpm/106 cells of triplicate samples were averaged, and incorporation was expressed
as percentage of untreated control. Variations between triplicate samples did notexceed 10%. , ADR; , AD 32; •¿�,0.1 fig; A. 1.0 fig; •¿�.5 fig.
JULY 1981 2747
Association for Cancer Research. by guest on September 1, 2020. Copyright 1981 Americanhttps://bloodcancerdiscov.aacrjournals.orgDownloaded from
A. Krishan et al.
Table 2Chromosomal damage ¡nCCRF-CEM cells exposed to ADR and AD 32
ControlADM0.01
/ig1.0jigAD-320.5
/ig1.0M10.0
/igDamaged/nor
mal30/256/1111/59/67/116/1Breaksand
gaps"0713111451Fragments0023410Translocations02134632
Number of metaphase plates with visibly evident chromosomal damage/plates with no gross visible damage. Chromosomal aberrations listed are the totalseen in the damaged plates.
0 Includes clear chromatid breaks (single and double) as well as clear
nonstained gaps in the chromatids.c Chromosomal fragments scattered in the metaphase plates.d Translocations involving one or both arms and often including multiradials.
U)
uu
u.o
(K
LJ
n
IO
IO
2C 4C 4C
RELfìTIVEflMOUNTOF DNRChart 3. DNA distribution histograms of CCRF-CEM cells exposed to various
concentrations of ADR and AD 32 for 24 hr. Control: A and E. ADR: 0.1 ng/ml(B); 1.0,ug/ml(C); 10/ig/ml (D). AD 32: 0.1 fig/ml (F); 1.0 fig/ml (G); 10(ig/ml(H).
ml (Chart 3H)], a part of the population is blocked in G2 whilethe rest of the population is still in Gì,presumably due to thedrug-induced inhibition of cell cycle traverse. Reincubation ofG2-blocked cells (as a result of incubation with AD 32 at 0.1^g/ml for 48 hr or 1.0 jug/ml for 24 hr), after repeated washingin drug-free medium, did not result in either resumption of cellcycle traverse or an increase in cell counts.
To evaluate the relation between the cytocidal effects of AD32 and the cell cycle position of CCRF-CEM lymphoblasts, log-phase cultures were synchronized by exposure to a double
block of IMPY (18) to obtain G,-early-S and mid-S cell popu
lations. Cultures exposed to a double block of 2.0 mw IMPYhad a 7% labeling index, and the incorporation of thymidinewas approximately 10% of the log-phase cultures. Flow cy-
tometry showed that most of the cell population (approximately90%) had the DNA content of d. Of the cells released fromthe block by washing and reincubation in fresh medium for 4hr, 90% were in DNA synthesis, as determined by flow cy-tometry and autoradiography. Details of this method have beenpublished before (18).
Cultures, in triplicate, of synchronized (Gì,S) populationswere exposed to various concentrations of ADR (0.5 and 1.0ftg/ml)and AD 32 (1.0, 2.0, and 6.0,ug/ml). After 1 hr exposureto the drug, cells were washed twice and reincubated in freshmedium for 24 hr. Cell counts, after trypan blue staining, weretaken to evaluate the cytotoxic effects of the drugs. Data inTable 3 show that, in cultures exposed to ADR, an approximately 2-fold higher cell kill was seen in S-phase cultures than
in cells exposed to the drug in Gìphase. In contrast, no majordifference was seen between cells exposed in either G, or Sphase to AD 32.
In another set of experiments, the cytotoxic effects of ADRand AD 32 on cells from log and plateau-phase cultures werecompared. As described earlier (15), plateau-phase cultureswere obtained by allowing CCRF-CEM cell cultures to grow toa saturation density of 3.5 x 106/ml. Pulse-labeling index in
these cultures was approximately 10% (with low grain count)and incorporation of thymidine into DNA was 8 to 10% of thecontrol, log-phase cultures. Data in Table 3 further show that,in cultures exposed to ADR, more cells are killed in log-phasethan in plateau-phase cultures. In contrast, cell counts in log-phase or plateau-phase cultures exposed to AD 32 were ap
proximately similar.In subsequent experiments, L1210 cells were processed in
an elutriator rotor to obtain different subpopulations based oncell density and mass. DNA distribution histograms were usedto identify various populations on the basis of their cell cycleposition. Subpopulations collected for the Gìfraction containedpredominantly cells with GìDNA content. The labeling index ofthis population was approximately 18% (compared to 60% inthe log-phase population) and obviously included some earlyS-phase cell. The S-phase population collected containedmostly cells with mid-S-phase DNA content and a labelingindex of 75%. The population collected for late S-(G?-M) had
Table 3Cytotoxic effects of ADR and AD 32 in relation to CCRF-CEM cell cycle
No. of dye-excluding cells (% of controls)3
G," Log" Plateau6
ADR0.5jig1.0
MOAD
32iMS2fig6
ng523480462820675139235250416249655567
Values represent mean of counts from 3 flasks; standard deviations wereless than 5%.
6 G,-early S cells. Labeling index, 7%.c Mid-S phase. Labeling index, 90%.d Log phase (1.4 x 10e cell/ml). Labeling index. 60%.8 Plateau phase (3.5 x 106 cells/ml). Labeling index, 10%.
2748 CANCER RESEARCH VOL. 41
Association for Cancer Research. by guest on September 1, 2020. Copyright 1981 Americanhttps://bloodcancerdiscov.aacrjournals.orgDownloaded from
ADR and AD 32 in Vitro
a labeling index of 55%, and DMA distribution histogramsrevealed that most of these cells were in late S and G2-M parts
of the cell cycle. Cells from these selected populations wereexposed to various drug concentrations for 1 hr, washed indrug-free medium, and plated in soft agar. Data in Chart 4
summarize the effect of various drug concentrations on thecolony-forming ability of these elutriated L1210 subpopulationsin soft agar. In cells exposed to ADR, a drug concentration-related loss of clonogenicity was evident in all 3 cell populations. However, no greater sensitivity of mid-S-phase cells as
compared to that of G, cells to ADR could be demonstrated.The cell population with late S-(G?-M) cells was slightly moresensitive (0.01 to 1 ^g/ml) than were the G, and mid-S populations. However, in cells exposed to ADR (10 fig/ml), the mid-S-phase cells were slightly more sensitive than the late S-(G?-
M) population.
Gl/ERRLY S PHRSE
10080804020O7I1
S-PHRSE
iWo(_>
«0u.
soo40t—
Z20u
oT^Ti1T1il^oUT
LRTE SXG2-M PHRSE
100•0co40to0rH
-1-i-52§0
0 J_-*-lII11o
—¿�o oooo-ooo
o o —¿�o
RDRIRMYCIN HD-32
(ug
Chart 4. Effect of ADR and AD 32 on the colony-forming ability of elutriatedL1210 subpopulations in soft agar. Bars, S.D.
In cells exposed to AD 32, no major cell cycle phase-relatedcytotoxicity could be demonstrated. Cells in late S-(G?-M) wereslightly more sensitive than were cells in mid-S and G,. Themid-S-phase cells were uniformly more resistant than were Gìand late S-(G2-M) populations.
In cells exposed to very high drug concentrations (10 /tg/ml), results were variable due to the small number of coloniesseen per plate. These experiments on elutriated cell populations with soft-agar colony-forming assays were repeated on 3
different occasions and, unlike the data (not based on soft agarassays) earlier reported by us and others, we could not demonstrate a cell cycle phase-related enhanced drug sensitivity
in L1210 cells to either ADR or AD 32 in this assay. In contrast,aliquots of d and S phase-elutriated subpopulations incubatedwith 1- and 10-/ig/ml doses of 1-/8-D-arabinofuranosylcytosine
for 1 hr before washing and plating in soft agar demonstrateda 2- to 3-fold higher sensitivity of the S-phase cells than that of
the G, cells.
DISCUSSION
The therapeutic superiority of AD 32 over ADR in experimental rodent tumor systems has been confirmed in our ownlaboratory and elsewhere (8, 21, 27). However, in vitro, AD 32is somewhat less active than ADR in inhibiting the growth ofcultured cells. The ID50 values presented in this report for 24-hr cultures of CCRF-CEM cells are consistent with previouslydetermined ID50values for 48-hr continuous cultures (8, 19). Incontrast to CCRF-CEM and LAZ-007 cells, where ADR was 2-to 3-fold more cytotoxic than AD 32, in L1210 cells, the ID50
values of the 2 drugs were approximately similar. This was alsoconfirmed in the soft-agar colony-forming assay for L1210
cells, when the number of colonies after treatment with AD 32were only 23 to 60% greater than for cells exposed to similarconcentrations of ADR.
However, despite its lower growth-inhibitory activity againstcultured CCRF-CEM cells, data presented in this report show
that AD 32, like ADR, produces chromosomal damage and asimilar inhibition of nucleic acid and protein synthesis, asmonitored by radiolabeled precursor incorporation. The valuesfor the inhibition of thymidine and uridine incorporation seenhere are in agreement with results from a parallel study (19)which included an analysis of anthracyclines in cell sonicatesand culture media of cells treated with ADR and AD 32. At 10times the 48-hr ID50 values for ADR and AD 32, sonicates of
cells incubated with AD 32 for 2 hr showed only trace amountsof ADR by high-sensitivity liquid Chromatographie assay, incomparison to ADR levels from ADR-treated cells (19).
AD 32 and ADR show comparable concentration and timedependency effects, and they produce a similar block at G2 incell cycle traverse. AD 32, however, does not show the cellcycle phase specificity or log-phase growth versus plateau-
phase growth sensitivity seen with ADR.In cells synchronized by exposure to IMPY and treated with
ADR or AD 32 in various phases of the cell cycle, we coulddemonstrate the enhanced selective sensitivity of S-phase cells
to the cytotoxic activity of ADR. However, a similar differentialsensitivity to AD 32 could not be demonstrated in these experiments. Similarly, the relative reduced sensitivity of plateau-phase cells to ADR (as compared to that of log-phase cells)
could not be clearly shown in cells exposed to AD 32.
JULY 1981 2749
Association for Cancer Research. by guest on September 1, 2020. Copyright 1981 Americanhttps://bloodcancerdiscov.aacrjournals.orgDownloaded from
A. Krishan et al.
Table4Comparisonol in vitro effects of ADR andAD-32Intracellular
transport show, temperature andtimedependent(1 7,20).Intracellular
fluorescence in live cells localized innucleiandon chromosomes (17).Fluorescent
staining of fixed nuclei andchromosomes(17).Binding
to isolated calf thymus and other DMA(22).Heterochromatincondensation innuclei.Interferes
(quenching) with binding of otherDNA-binding
fluorochromes(16).Extensivechromosomaldamage.0Inhibition
in nucleic acid (RNA, DMA)synthesis.Selectivesensitivity of log-phase vs.plateau-phaseculture."Cell
cycle phase-specificcytotoxicity."
A. Krishan, unpublishedobservations.Present
observations.ADRYesYesYesYesYesYesYesYesYesYesAD
32NoNoNoNoNoNoYesYesNoNo
In soft-agar colony-forming assays for L1210 cells, we havenot been able to confirm the greater sensitivity of the S-phase
cells (as compared to that of Gt cells) as reported earlier by usand other workers to either ADR or AD 32. Our earlier studieswere based on CCRF-CEM cells and most of the other studies
have been based on Chinese hamster ovary cells or HeLa cells.It is possible that the special conditions of growth in soft agarmay allow for better repair of drug-induced damage and thusnot show the cell cycle phase-related sensitivity shown in other
systems. Experiments are under way to test if a similar lack ofcell cycle phase-related cytotoxicity can be demonstrated in
other cell culture systems with the soft agar assay.In conclusion and as listed in Table 4, the present study and
earlier observations indicate that, in spite of some differencesin intracellular transport, localization of intracellular fluorescence, and cell cycle phase-related cytotoxicity, ADR and AD
32 display a variety of similar effects on macromolecular synthesis and chromosomal structure. These observations suggestthat AD 32 and ADR may have similar mechanisms of action.However, because of its structure, AD 32 is not able to bind toDNA in the same manner as does ADR. If, in fact, AD 32 doesnot give rise to biologically significant levels of ADR in vitro (8,19) or in vivo (6, 9, 11), then the widely accepted hypothesisbased on the intercalation of ADR and DNA is not adequate toexplain the biological effects of ADR, and the ADR mechanismof action requires modification. As shown by Bachur et al. (1),a mechanism of action based on generation of "site-specificfree radicals" may provide a better explanation for the mode
of action of quinone-containing anticancer agents like ADR and
AD 32.
REFERENCES
1. Bachur, N. R., Gordon, S. L, and Gee. M. V. A general mechanism formicrosomal activation of quinone anticancer agents to free radicals. CancerRes., 38. 1745-1750, 1978.
2. Bagwell. C. B. Theory and application of DNA histogram analysis. Ph.D.dissertation, University of Miami, Coral Gables. Fla.. 1979.
3. DiMarco, A., and Arcamene, F. DNA complexing antibiotics: daunomycin.Adriamycin, and their derivatives. Arzneim-Forsch.. 25: 368-375, 1975.
4. DiMarco, A., and Lenaz, L. Daunomycin and Adriamycin. In: J. F. Hollandand E. Frei, III (eds.), Cancer Medicine, pp. 826-835. Philadelphia: Lea andFebiger, 1973.
5. Henderson, I. C., Billingham, M., Israel. M., Krishan, A., and Frei, E., III.Comparative cardiotoxicity studies with Adriamycin (ADR) and AD 32 inrabbits. Proc. Am. Assoc. Cancer Res.. Õ9:158, 1978.
6. Israel, M., Garnick, M. G., Pegg. W. J., Blum, R. H., and Frei, E., III.Preliminary pharmacology of AD 32 in man. Proc. Am. Assoc. Cancer Res../ 9. 160, 1978.
7. Israel, M.. Karkowsky, A. M., and Pegg, W. J. Pharmacological studies withradiolabeled W-trifluoroacetyladriamycin-t4-valerate (AD 32). Comparisonof total fluorescence and radioactivity in mouse serum and urine. CancerChemother. Pharmacol., 4: 79-82, 1980.
8. Israel, M., Modest, E. J., and Frei, E., III. N-Trifluoroacetyladriamycin-14-valerate, an analog with greater experimental antitumor activity and lesstoxicity than Adriamycin. Cancer Res., 35: 1365-1368, 1975.
9. Israel, M., Pegg, W. J.. and Wilkinson, P. M. Urinary anthracycline metabolites from mice treated with Adriamycin and W-triftuoroacetyladriamycin-14-valerate. J. Pharmacol. Exp. Therap., 204. 696-701. 1978.
10. Israel, M., Wilkinson. P. M.. Pegg, W. J., and Frei, E.. III. Hepatobiliarymetabolism and excretion of Adriamycin and W-trifluoroacetyladriamycin-14-valerate in the rat. Cancer Res., 38. 365-370, 1978.
11. Karkowsky, A. M.. and Israel, M. Serum levels and tissue distribution studiesin mice with radiolabeled AD 32. Proc. Am. Assoc. Cancer Res., 19: 157,1978.
12. Kim, S. H., and Kim, J. H. Lethal effect of Adriamycin on the division cycleof HeLa cells. Cancer Res.. 32. 323-325, 1972.
13. Kobayashi, U.. and Maudsley. D. V. Recent advances in sample preparation.In: P. Stanley and B. Scoggins (eds.). Liquid Scintillation Counting—RecentRedevelopments, pp. 189-205. New York: Academic Press, Inc., 1974.
14. Krishan, A. Rapid flow cytofluorometric analysis of mammalian cell cycle bypropidium iodide staining. J. Cell Biol., 66. 188-193. 1975.
15. Krishan, A., and Frei, E., III. Effect of Adriamycin on the cell cycle traverseand kinetics of cultured human lymphoblasts. Cancer Res., 36: 143-150,1976.
16. Krishan, A., Ganapathi, R. N., and Israel, M. The effect of Adriamycin andanalogs on the nuclear fluorescence of propidium iodide stained cellsCancer Res., 38: 3656-3662, 1978.
17. Krishan, A., Israel, M.. Modest, E. J., and Frei, E., III. Differences in cellularuptake and cytofluorescence of Adriamycin and N-trifluoroacetyladriamycin-14-valerate. Cancer Res., 36: 2108-2110, 1976.
18. Krishan, A., Paika, K. D., and Frei, E.. III. Cell cycle synchronization ofhuman lymphoid cells in vitro by 2,3-dihydro-1H-imidazo(1,2-o)pyrazole.Cancer Res.. 36: 138-142. 1976.
19. Lazarus, H., Yuan, G.. Tan, E., and Israel. M. Comparative inhibitory effectsof Adriamycin. AD 32, and related compounds on in vitro cell growth andmacromolecular synthesis. Proc. Am. Assoc. Cancer Res., 19: 159, 1978.
20. Meriwether, W. D.. and Bachur, N. R. Inhibition of DNA and RNA metabolismby daunorubicin and Adriamycin in L1210 mouse leukemia. Cancer Res..32. 1137-1142, 1972.
21. Parker, L. M., Hirst, M., and Israel, M. N-Trifluoroacetyladriamycin-14-val-erate: additional mouse antitumor and toxicity studies. Cancer Treat. Rep ,62: 119-127, 1978.
22. Sengupta. S. K., Seshadri, R.. Modest, E. J., and Israel, M. ComparativeDNA-binding studies with Adriamycin (ADR), W-trifluoroacetyladriamycin-14-valerate (AD 32). and related compounds. Proc. Am. Assoc. Cancer Res.,77: 109, 1976.
23. Sheck, L. E.. Muirhead, K. A., and Horan, P. K. Evaluation of the S phasedistribution of flow cytometric DNA histograms by autoradiography andcomputer algorithms. Cytometry. 1: 109-117, 1980.
24. Silvestrini, R.. Gambarucci. C., and Dasdia. T. In vitro biological activity ofAdriamycin. Tumori, 56: 137-148, 1970.
25. Skovsgaard, T., and Nissen, N I. Adriamycin. an antitumor antibiotic: areview with special reference to daunomycin. Dan. Med. Bull., 22: 62-73,1975.
26. Tobey. R. A. Effects of cytosine arabinoside, daunomycin, mithramycin,azacytidine. Adriamycin, and camptothecin on mammalian cell cycle traverse. Cancer Res., 32: 2720-2725, 1972.
27. Vecchi, A., Cairo. M., Mantovani, A., Sironi, M., and Spreafico, F. Comparative antineoplastic activity of Adriamycin and N-trifluoroacetyladriamycin-14-valerate. Cancer Treat. Rep., 62. 111-117. 1978.
28. Vig, B. K. Chromosome aberrations induced in human leukocytes by theantileukemic antibiotic Adriamycin. Cancer Res., 31: 32-38, 1971.
29. Whang-Peng. J., Leventhal. B., Adamson. J. W., and Perry S. The effect ofdaunomycin on human cells in vivo and in vitro. Cancer (Phila.), 23: 113-121, 1969.
2750 CANCER RESEARCH VOL. 41
Association for Cancer Research. by guest on September 1, 2020. Copyright 1981 Americanhttps://bloodcancerdiscov.aacrjournals.orgDownloaded from