[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

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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

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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

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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

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1941.0MO/

ml30

815.0

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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/

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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.

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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.

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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%.

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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.

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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.

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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.

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