cytotoxicity of daunorubicin in trisomic (+21) human fibroblasts: relation to drug uptake and cell...

Cell Biology International 2001, Vol. 25, No. 2, 157–170doi:10.1006/cbir.2000.0583, available online at http://www.idealibrary.com on

CYTOTOXICITY OF DAUNORUBICIN IN TRISOMIC (+21) HUMAN FIBROBLASTS:RELATION TO DRUG UPTAKE AND CELL MEMBRANE FLUIDITY

MARIA PRZYBYLSKA, ANETA KOCEVA-CHYŁA, BŁAZrEJ ROu ZGA and ZOFIA JOu ZuWIAK

Department of Thermobiology, Institute of Biophysics, University of Łodz, 12/16 Banacha St, 90-237 Łodz, Poland

Received 11 November 1999; accepted 13 June 2000

The influence of daunorubicin (DNR) on survival of human normal (S-126) and trisomic, withrespect to chromosome 21 (T-164; S-240), skin fibroblasts and some parameters related to it,such as intracellular drug accumulation, distribution and interaction with cell membrane, werestudied. The in vitro growth-inhibition assay indicated that DNR was less cytotoxic for trisomicthan for normal cells. Comparison of kinetic parameters and intracellular distribution of thiscompound showed that the uptake and the amount of intracellular free DNR were greater innormal than in trisomic cells. Contrary to this, there were no significant differences between theamount of DNA-bound drug in both types of cells. TMA-DPH and 12-AS fluorescenceanisotropy measurements demonstrated that DNR decreased lipid fluidity in the inner hydro-phobic region of plasma membrane in both cell types, but did not influence the fluidity of theouter surface of membrane. We conclude that fibroblasts derived from individuals affected withDown’s syndrome are better protected from the damage induced by DNR than normal cells.

� 2001 Academic Press

K: Down’s syndrome; fibroblasts; daunorubicin; cytotoxicity; intracellular accumulation; cellmembrane fluidity; TMA-DPH; 12 AS.

To whom correspondence should be addressed: Dr Maria Przybylska,Department of Thermobiology, Institute of Biophysics, Universityof Łodz, 12/16 Banacha St, 90-237 Łodz, Poland. E-mail:[email protected]

INTRODUCTION

Trisomies are the most common genetic disordersin human conceptions and have been describedfor each of 23 chromosomes (Heim and Mitelman,1986; Mitelman et al., 1991). Sole karyotypeaberrations, with respect to chromosomes 8, 10,12, 13, 15, 16, 18 and 21, are especially frequent(Nicolaidis and Petersen, 1998). Approximatelyhalf of all early spontaneous abortions are due tochromosome abnormalities, most of which are tri-somies (Hassold et al., 1980). The gains of singleadditional chromosomes are genomic aberrationspredominantly found in haematological malignan-cies (Haglund et al., 1997; Schoch et al., 1997;Kojima et al., 1998; Luno et al., 1998; Ma et al.,1998), although, some of them are also detected inpatients suffering from different kinds of solid

1065–6995/01/020157+14 $35.00/0

tumours. Trisomy 7 is found in various tumours ofbladder, kidney, lung, colon, brain and thyroidgland; trisomy 10 is observed in adenocarcinoma ofthe kidney and uterus; and trisomy 12 is indicatedin various cancers of ovary and the uterus(Mitelman et al., 1991; Herrmann and Lalley,1992). These facts suggest the significance of aneu-ploidy abnormalities in the pathogenesis of someneoplastic disorders (Berger et al., 1982; Heim andMitelman, 1986; Haas and Seyger, 1993). Thecurrent opinion on the pathogenesis of trisomies inmalignant process is based on the hypothesis thatthe gene dosage effect promotes the genomic in-stability. It has been considered as an initial stepin the formation of a neoplastic cell populationand more complex abnormalities observed dur-ing tumour progression (Haas and Seyger, 1993;Haglund et al., 1997).

The best-known example of an autosomal aneu-ploidy is trisomy of the twenty-first chromosome,which causes Down’s syndrome (DS). The overallincidence of this abnormality is approximately 1 in

� 2001 Academic Press

158 Cell Biology International, Vol. 25, No. 2, 2001

700 live births and the social trends progressivelytend to augmentation of the frequency of thisdisease occurrence, due to its strong associationwith advanced maternal age (Polani et al., 1976;Shapiro, 1994; Mueller and Young, 1998). In ad-dition to mental deficiency and other pathologicalconsequences, individuals with constitutional com-plete or mosaic trisomy 21 have pronounced riskof developing haematological neoplasms (Fersteret al., 1986; Iselius et al., 1990; Bunin et al., 1991;Robinson, 1992). Trisomy 21 is a common featurein different kinds of leukaemias (acute lympho-blastic leukaemia, acute nonlymphoblastic leukae-mia, megakaryoblastic leukaemia) and in transientmyeloproliferative disorder (Fong and Brodeur,1987; Iselius et al., 1990). Down’s syndrome isespecially frequently associated with childhoodcancer (Ragab et al., 1991; Lampert et al., 1992). Inthe case of affected children, the risk of leukaemiais increased about ten- to 20-fold compared to thatone observed in normal populations (Fong andBrodeur, 1987). About 3% of children with leukae-mia have also Down’s syndrome. Although, acutelymphoblastic leukaemia is the most frequent child-hood leukaemia in general population (about 85%of total cases of leukaemias diagnosed in U.S.A.)(Miller et al., 1994) the majority of childrenaffected with Down’s syndrome and leukaemiahave acute myelogenous leukaemia (AML) (65%)(Mertens et al., 1998). Within the broad category ofacute non-lymphoblastic leukaemias (ANLL), therelative risk for a rare subtype, the megakaryo-blastic leukaemia, is very much greater: half ofall cases of the megakaryoblastic leukaemia haveoccurred in children with DS (Lampert et al.,1992). The incidence of other cancers amongchildren with DS is unremarkable, but there is anevidence that males have an increased risk ofmalignant testicular germ cell tumours (Bergeret al., 1982; Lampert et al., 1992).

The widespread use of anthracycline antibioticsin clinical treatment of a broad spectrum of can-cers, for over 30 years, has stimulated intensiveinvestigation of the mechanism of their potentantineoplastic activities. Despite importantprogress in the understanding of the cell deathprocess induced by these drugs, the molecular basisfor their essential anticancer activity is not wellestablished. In this context, the elucidation of thespecific response of trisomic cells to anticancerdrugs is of special interest.

For this purpose we selected a representativemember of anthracycline antibiotics—dauno-rubicin (DNR). Daunorubicin is one of the mostwidely used antineoplastic drug, especially active

against the haematopoietic malignancies such as:acute lymphocytic and acute myelogenous leukae-mias, Hodgkin’s and non-Hodgkin’s lymphomas,multiple myeloma, carcinomas of the breast, lung,ovary, stomach and thyroid and various childhoodmalignancies (Doroshow, 1996; Hortobagyi, 1997).Generally, it is postulated that the molecular eventsresponsible for DNR efficacy include: intercalativeDNA binding leading to blocked transcription(Chaires, 1995; Leng et al., 1996; Wang et al.,1998), targeting to specific enzymes, such as topoi-somerase II (Capranico et al., 1990), mediatingDNA stand breaks and participation inredox cycling leading to free-radical generation(Goodman and Hochstein, 1977; Powis, 1989;Malisza et al., 1996; Otake et al., 1997; Taatjeset al., 1997). The majority of data support thehypothesis that plasma membrane is one of thecellular structures, responsible for many cancercell properties, such as invasiveness and growthfeatures, and an important target for DNR anti-neoplastic activity (Prosperi et al., 1985;Constantinides et al., 1990; Arancia and Donelli,1991; Daoud, 1992; Marcocci et al., 1992; Escribaet al., 1995; Leibovici et al., 1996; Malisza et al.,1996).

Taking into account the fact that the cytotoxiceffect of DNR is, at least partly, mediated byreactive oxygen species, it may be expected that theresult of its cytotoxic action will be different in thecase of cells derived from individuals suffering fromDown’s syndrome, relatively rich in superoxidedismutase (SOD-1) (EC: 1.15.1.1), an antioxidantenzyme encoded on the distal segment of the 21chromosome (Benson 1975; Kedziora and Bartosz,1988).

Thus, the aim of our study was the investigationof the effect of daunorubicin on the survival oftrisomic (+21) cells compared to normal ones aswell as the estimation of some parameters relatedto it, such as uptake dynamic and interaction withcell membrane. For this purpose, as a convenientmodel system, human normal (S-126) and trisomic,with respect to chromosome 21, (T-164, S-240)fibroblast lines were used.

MATERIAL AND METHODS

Drugs and chemicals

Daunorubicin (cerubidine) was obtained fromLaboratoire Roger Bellon (Neuilly Sur Seine,France), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and dimethyl sulfoxide

Cell Biology International, Vol. 25, No. 2, 2001 159

(DMSO) were purchased from Sigma ChemicalCo. (St Louis, MO, U.S.A). Fluorescenceprobes: 1-(4-trimethyl-ammoniumphenyl)-6-phenyl-1,3,5-hexatriene) (TMA-DPH) was from Mol-ecular Probes (Eugene, OR, U.S.A.) and 12-(9-anthroyloxy)-stearic acid (12-AS) was from SigmaChemical Co. Eagle’s Minimal Essential Mediumwas supplied by Sera and Vaccines Factory(Lublin, Poland) and Triton X-100 was obtainedfrom POCh (Gliwice, Poland). Calf serum andlactoalbumin hydrolysate were from Gibco BRL(Edinburgh, Scotland). Gentamycin was purchasedfrom Biochemie Gesellschaft m.b.H. (Vienna,Austria).

Cell lines and culture

Human fibroblasts derived from skin of individualsaffected with Down’s syndrome (T-164 cell line (47,XX, +21, fetus) and S-240 cell line (47, XY, +21,21 year)), and from skin of a normal donor (S-126cell line (46, XY 26 year)) were obtained fromtissue bank of the Centre of Child Health (Warsaw,Poland). Cell lines utilised during experiments wereregularly tested to ensure they were free of con-taminants and infections and re-established fromfrozen stocks at periods of 3-4 weeks to avoid‘phenotypic drift’. Cells were routinely grown as amonolayer in tissue culture flasks (Costar (D.Dutscher, France) or Nunc (Naperville, IL,U.S.A.)), in a humidified atmosphere of 5% CO2at 37�C in Eagle’s Minimal Essential Mediumenriched with 10% calf serum and 10% lactoalbu-min hydrolysate. Gentamycin was added at a con-centration of 0.0005%. For the experiments,exponentially growing cells between the 5th and15th passage were used.

Cytotoxicity assays

In vitro sensitivity assay to daunorubicin was per-formed in 96-well flat-bottomed plates (Costar) byusing the standard MTT colorimetric methodbased on the ability of the mitochondrial dehydro-genases of metabolically viable cells to reduce thetetrazolium salt (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) to blue formazanproduct (Carmichael et al., 1987). For this purpose,after trypsinization and cell counting, an equalnumber of fibroblasts (104) was seeded in each wellin 0.2 ml of culture medium. After an overnightincubation, 0.05 ml of DNR solution (at variousconcentrations) was added and cells were incubatedfor 2 h (37�C, 5% CO2). The same volume ofsodium phosphate buffered saline (PBS) was added

to control samples. After the desired time, mediumcontaining DNR (or PBS in the case of controlsamples) was removed, replaced by fresh mediumand cells were allowed to grow for 3 days (37�C,5% CO2). Then, 0.05 ml of MTT solution in PBS(6�10�3 mol/dm3) were added to each welland incubation was continued for a further 4 h.Subsequently, culture medium was removed andthe resulting formazan crystals were solubilized in0.1 ml of DMSO. The extend of MTT reduction toformazan within cells was quantitated by measure-ment of absorbance at 570 nm using microplatereader (Awareness Technology Inc, Palm City, FL,U.S.A.). The percentage of surviving drug-treatedcells in relation to control (non-treated cells) wasdetermined. The IC50 parameter, defined as drugconcentration that reduced cell growth to 50%compared with the control cells, was calculatedfrom linear transformation of the dose–responsecurves.

Drug uptake and intracellular distribution

The quantitative evaluation of DNR uptake bycells was measured essentially according to themethod elaborated by Andreoni and co-workers(1994, 1996). For this purpose, an equal number ofcells (106) in 3 ml of culture medium were platedinto series of 35-mm Petri dishes. After an over-night incubation (37�C, 5% CO2) growth mediumwas removed and replaced by the DNR solution(3�10�6 mol/dm3) in PBS. An equal volume ofPBS was added to control samples. Dishes contain-ing solution of drug (3�10�6 mol/dm3) withoutcells were used as references for initial drug concen-tration. At various times of exposure to the drug,medium was sucked up from dishes and centrifuged(600�g, 5 min). The amount of drug in mediumwas estimated using an LS-5B Perkin Elmerfluorescence spectrometer (Perkin Elmer Ltd.,Beaconsfield, Buckinghamshire, U.K.). The emis-sion of the samples was measured at 595 nm uponan excitation at 488 nm. The amount of DNRuptaken by fibroblasts was determined as follows.Cell monolayers were washed gently three timeswith 2 ml of PBS. Subsequently, 3 ml of culturemedium containing 0.05% Triton X-100 was addedto dishes to permeabilize cell membranes. After15 min, medium was transferred to a fluorimetriccuvette and the fluorescence intensity at 595 nm(�ex=488 nm) was measured. The content of freedrug and drug bound to DNA was determinedaccording to the assumptions that under this con-dition the fluorescence of anthracycline mol-ecules bound to cellular DNA is completely


quenched (Mankhetkorn et al., 1996; Tarasiuket al., 1989) and Triton X-100, at applied concen-tration, is not able to release daunorubicin fromits intercalative site in DNA molecules (Tarasiuket al., 1989; Frezard and Garnier-Suillerot,1991a,b; Mankhetkorn et al., 1996). The amount ofdrug in extracellular medium and associated withthe cells was calculated from the standard curve,representing the relationship between drug concen-tration and its fluorescence intensity.

For estimation of the DNR transport parametersa simple model of transport kinetics, involvingonly three compartments: (M)—extracellularmedium containing the M amount of drug; (F)—intracellular compartment containing the Famount of relatively free drug and (B)—containingthe B amount of DNR bound to cellular DNA,were used. The intracellular amount of drug(C=B+F) was calculated from the equation:

C=Mtot�Mt

where: Mtot represents the total amount of drug towhich cells were initially exposed and Mt is theamount of DNR in external medium at differenttime of incubation.

Values representing the intracellular amount ofdrug were fitted to third-order polynomials (deter-mination coefficients (R2) for plots were always>0.95). The initial velocity of DNR uptake (It=0)was calculated as a value of a first derivative of thecurve representing time-dependence of DNR trans-port. Uptake rate constants of DNR transportwere calculated in accordance with the assumptionthat DNR uptake occurred on the basis of a firstorder equation.

It=0=kin · Mtot

where: kin is the influx rate constant. The amountof drug taken up by cells was then calculated fromequation:

ln Mtot�ln Mt=kin · t

The transport parameters for drug excluded bycells (kout and Et=0) were calculated in analogousmanner from the curves represented the time de-pendence of the values obtained by the subtractionof the intracellular amount of drug (C) from theamount of drug taken up by cells (U) at the sameincubation time.

TMA-DPH fluorescence quenching

The association of daunorubicin with the plasmamembrane of intact cells was investigated in-

directly, by measurement of the fluorescence reson-ance energy transfer (Mulder et al., 1993). In thisstudy, the plasma membrane fluorescent probeTMA-DPH was used. Transfer of energy can beachieved from TMA-DPH to DNR due to theoverlap between the emission spectrum of TMA-DPH and the excitation spectrum of DNR. Thecells (2�105), resuspended in 1.8 ml of PBS, wereincubated with TMA-DPH (extracellular concen-tration 1.5�10�6 mol/dm3), at 37�C, for 30 min.Then, DNR was added at a final concentration of10�4 mol/dm3. Rapid initial intracellular TMA-DPH fluorescence quenching was determined byfitting of the data to logarithmic trend line andextrapolating to a time as 1 s. Second phasequenching (t=2 min) was determined after reachingsteady state TMA-DPH fluorescence intensity.TMA-DPH fluorescence quenching was expressedas a percentage of the intracellular steady-stateTMA-DPH fluorescence that had been reachedbefore addition of DNR.

Membrane fluidity

To determine the effect of daunorubicin on thecell membrane fluidity, equal number of cells(3�105 cells/ml) suspended in 1.8 ml of culturemedium were placed into the set of test tubes.Then, 0.05 ml of drug solutions, at various con-centrations (2–100�10�6 mol/dm3) were added.Samples containing fibroblasts suspended in me-dium without drug were used as controls. Afterincubation (2 h in humidified atmosphere of 5%CO2, at 37�C) samples were centrifuged (600�g,5 min), the supernatant was drawn off and thepellet was resuspended in 1.8 ml PBS. To evaluatemembrane fluidity, cell suspensions were incubatedwith fluorescence probes TMA-DPH or 12-AS at20�C for 10 and 15 min, respectively. Measure-ments were made using an LS-5B Perkin ElmerSpectrofluorimeter. The excitation and the emis-sion wavelengths were �ex=358 nm and �em=428 nm for TMA-DPH and �ex=360 nm and�em=471 nm for 12-AS. Fluorescence anisotropywas calculated according to the equation (Van deMeer, 1988).

r=(Ivv�Ivh G)/(Ivv+2IvhG),

where: Ivv and Ihv represent the components of thelight intensity emitted, respectively, parallel andperpendicular to the direction of the verticallypolarized excitation light and G is the correctionfactor (G=Ihv/Ihh).


Studies on membrane fluidity performed bymeasuring the emission anisotropy of the fluor-escent probes, TMA-DPH and 12-AS, yieldedinformation about the rigidity of the cell membranenear the lipid polar heads (Kuhry et al., 1985) andinside the hydrophobic lipid core respectively(Podo and Blasie, 1977).

Statistical analysis

Data were analysed by one-way analysis of vari-ance (ANOVA), and P<0.01 was considered to bestatistically significant (Zar, 1984). All values wereexpressed as mean�standard deviation of six ormore separate experiments repeated six to seventimes.

RESULTS

Cytotoxicity assays

The growth-inhibition assay indicated that the ex-posure of human fibroblasts to DNR induced theprogressive diminishing of cell survival in a doseresponse manner. Analysis of survival curves ob-tained for normal (S-126) and trisomic (T-164,S-240) cell lines (Fig. 1), demonstrated that thesensitivity of fibroblasts derived from DS individ-uals to DNR was lower than the sensitivity ofnormal cells. Comparison of the IC50 values(S-240—6.9�0.8 �; T-164—7.5�0.9 �; S-126—4.2�0.5 �) indicated that the antiproliferative

effect of DNR is, on an average, about 70% greaterfor normal than for trisomic cells.

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Fig. 1. Survival curves of normal (S-126, �) and trisomic(S-240, � and T-164, �) human fibroblasts treated withdaunorubicin. The growth-inhibition assay was performedusing the standard MTT colorimetric method. Data are ex-pressed as mean�standard deviation for seven independentexperiments.

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Fig. 2. Time-dependence of DNR taken up (�) and DNRremoved (�) by human normal (S-126, A) and trisomic(S-240, B; and T-164, C) fibroblasts. (): amount of drug inexternal medium. (�): amount of drug associated with cells.Data are expressed as mean�standard deviation for sixindependent experiments.

Drug uptake and intracellular distribution

The studies of the dynamic of DNR transportthrough the cellular membrane, estimated in-directly by measurement of the fluorescence emis-sion intensity, showed the difference in cellularuptake of this compound by normal and trisomiccells. The analysis of curves representing the time-dependence of amount of drug taken up (U) andamount of drug excluded by cells (E) (Fig. 2) andthe comparison of the kinetic parameters, derivedfrom them (Table 1), indicated that the amount of


drug taken up by cells, within 60 min of incubation,was considerably greater (one-way ANOVA:P<0.01) for normal than for trisomic cells(S-240—6.05�0.14 nmol/106 cells; T-164—5.55�0.30 nmol/106 cells; S-126—7.82�0.07 nmol/106 cells). Comparison of the values representingthe amount of DNR removed by fibroblasts within60 min of incubation (S-240—2.02�0.33 nmol/106 cells; T-164—1.74�0.18 nmol/106 cells; S-126—2.37�0.28 nmol/106 cells) suggests that efflux ofDNR is slightly greater (not statistically significant)for normal fibroblasts than for trisomic ones (Table1). Analysis of the intracellular distribution ofDNR (Fig. 3) showed that the amount of intra-cellular free DNR is greater (one-way ANOVA:P<0.01) for normal than for trisomic cells(S-240—1.89�0.24 nmol/106 cells; T-164—1.84�0.20 nmol/106 cells; S-126—3.67�0.25 nmol/106 cells) (Fig. 3). Contrary to this, no statisticallysignificant difference in the amount of DNR boundto DNA, for normal and trisomic fibroblasts,was observed (S-240—0.63�0.12 nmol/106 cells;T-164—0.61�0.13 nmol/106 cells; S-126—0.78�0.15 nmol/106 cells) (Fig. 3).

TMA-DPH fluorescence quenching

The assay of the TMA-DPH fluorescence quench-ing caused by the fluorescence resonance energytransfer from the fluorescent probe, TMA-DPH, toDNR demonstrated that the incorporation ofDNR into the outer leaflet of the plasma mem-brane of intact cells was very fast and achieveda plateau within the first minute of experiment(Fig. 4). The comparison of the diminution in theTMA-DPH fluorescence emission intensity indi-cated that the rapid initial quenching (time=1 s)

(S-240—8.87�1.32%; T-164—7.24�1.34%; S-126—14.09�2.39%) as well as the second phasequenching (time=2 min) (S-240—16.26�2.59%;T-164—10.46�2.78%; S-126—24.87�3.25%) werelower (one-way ANOVA: P<0.01) for both trisomicfibroblast lines than for normal cells.

Table 1.Transport parameters of daunorubicin in human normal (S-126) and trisomic (S-240, T-164)

fibroblasts

S-126 S-240 T-164

kin [min�1] 0.0338�0.0009 0.0186�0.0008* 0.0160�0.0014*It=0 [nmol/min/106 cells] 0.305�0.008 0.167�0.007* 0.144�0.012*Ut=60 [nmol/106 cells] 7.82�0.07 6.05�0.14* 5.55�0.30*kout [min�1] 0.0078�0.007 0.0074�0.0008 0.0061�0.0005Et=0 [nmol/min/106 cells] 0.070�0.0063 0.067�0.0070 0.055�0.0038Et=60 [nmol/106 cells] 3.37�0.28 3.22�0.33 2.77�0.18

Kin—influx rate constant; It=0—initial influx, Ut=60—drug taken up by cells within 60 min;Kout—efflux rate constant; Et=0—initial efflux; Et=60—drug removed by cells within 60 min. Data areexpressed as mean�standard deviation for six independent experiments.*Statistical significant difference (one-way ANOVA: P<0.01) compared with the S-126 cell line.

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Fig. 3. Cellular distribution of daunorubicin in normal (S-126)and trisomic (S-240 and T-164) human fibroblasts. : amountof DNR in external medium; �: amount of cellular free DNR;: amount of DNR bound to DNA. Data are expressed asmean�standard deviation for six independent experiments.*Significantly different when compared to values obtained forthe S-126 cell line (P<0.01).

Cell membrane fluidity of intact fibroblasts

The comparison of anisotropy values obtained byusing fluorescent probes—TMA-DPH and 12-AS—indicated that the trisomic fibroblasts hadsubstantially (P<0.01) lowered fluidity near the


outer surface lipid polar heads (S-240—r=0.295�0.009; T-164—r=0.293�0.008; S-126—r=0.272�0.007) than control cells (Fig. 5). How-ever, the fluidity at the inner, hydrophobic lipidregion of the cell membrane (S-240—r=0.134�0.009; T-164—r=0.132�0.011; S-126—r=0.129�0.010) was nearly the same for both celltypes (Fig. 6).

Effect of DNR on membrane fluidity

The fluorescence anisotropy of the fluorescentprobe 12-AS indicated that the incorporation ofDNR into the plasma membrane of intact cellsdecreased the membrane fluidity in the hydro-phobic region of the cell membrane of normal aswell as of trisomic cells, especially for drug concen-trations above 5�10�6 mol/dm3 (Fig. 6).

For the highest DNR concentration (10�4 mol/dm3) the fluorescence anisotropy (r) of 12-ASincreased from about 0.133 in untreated trisomiccells and from 0.129 in the case of normal cells toabout 0.240 for all three cell lines. Contrary to this,the TMA-DPH fluorescence anisotropy valuesdemonstrated no statistically significant influenceof DNR on the membrane fluidity near the outersurface lipid polar heads, even at the highest testeddoses (Fig. 5).

Time (min)

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Fig. 4. TMA-DPH fluorescence quenching induced by dauno-rubicin in normal (S-126, �) and trisomic (S-240, �; andT-164, �) fibroblasts. Data are expressed as mean�standarddeviation for six independent experiments.

DISCUSSION

The additional chromosome 21 in subjects affectedwith Down’s syndrome results an augmentationof the catalytic activity of Cu–Zn superoxide

dismutase (EC: 1.15.1.1), an enzyme coded ondistal segment of this chromosome (Benson, 1975;Kedziora and Bartosz, 1988; Gerli et al., 1990).Elevated levels of SOD-1, associated with an adap-tive rise in the activity of the H2O2 scavengingenzymes, glutathione peroxidase (GSH-Px) (EC:1.11.1.9), catalase (EC: 1.11.1.6) and glutathionereductase (EC: 1.6.4.2), increased antioxidative de-fence in DS cells, resulting in reduced generation ofsuperoxide anions and hydroxyl radicals (Kedzioraet al., 1990; Pastor et al., 1998). Alteration of thebalance of reactive oxygen species (ROS) may be akey point in the pathogenesis of DS as well as in thepotentially altered response of DS cells to externalsources of free radicals (Kedziora and Bartosz,1988; Brugge et al., 1992). Since the cytotoxic effectof daunorubicin is partly mediated by ROS gener-ation (Malisza et al., 1996; Otake et al., 1997;Taatjes et al., 1997), its cytotoxicity could bealtered in DS cells.

Although biochemical disturbances of the ROSmetabolism revealed in DS patients have beendescribed (Gerli et al., 1990; Chaires et al., 1995;Leng et al., 1996; Pastor et al., 1998; Wang et al.,1998), the role of elevated SOD-1 levels in sensitiv-ity of different types of trisomic (+21) cells to thevarious physical and chemical agents is still underdiscussion. Rozga and co-workers (1990, 1994)demonstrated significantly lower sensitivity oftrisomic cell lines to carminomycin compared todiploid non-DS cells. The same DS cell lines,however, were more sensitive to �-radiation(Kedziora et al., 1986), suggesting more unrepaireddouble-stranded DNA breaks, and consequent celldeath, as found by Otsuka et al. (1985), withX-rays. This implies a decreased DNA-repaircapacity in DS subjects (Raji et al., 1998).

Doroshow (1986) reported protective effect ofantioxidative SOD-1, catalase and a PZ51 (com-pound of GSH-Px-like activity) in MCF-7 cellstreated with adriamycin (ADR). Sublines ofMCF-7 cells resistant to ADR had increasedGSH-Px activity (Batist et al., 1986), but no in-creased levels of antioxidant enzymes were detectedin ADR-resistant human lung GLC4 (Meijer et al.,1987) and murine lymphoma cell lines (Ramu et al.,1988). No difference in ADR-sensitivity betweenCHO cells sublines with increased levels of GSH,SOD-1, catalase and GSH-Px enzymes and theparent CHO line was found after correction fordifferential drug uptake (Keizer et al., 1988). Onthe other hand, while SOD-1 is generally elevatedin DS cells, another closely-related antioxidativeenzyme, Mn-SOD (EC: 1.15.1.2), was considerablydecreased (Nohl and Jordan, 1983).


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Fig. 5. Influence of daunorubicin on fluorescence anisotropy of TMA-DPH in normal (S-126, ) and trisomic (S-240, �; andT-164, ) human fibroblasts. Data are expressed as mean�standard deviation for eight independent experiments.

Reports on DNR cytotoxicity vary with the celltype being tested (Gollapudi et al., 1994; Seidelet al., 1995; Houba et al., 1998). Differences intoxicity could be due to alterations in severalother enzymes involved in DNR metabolism anddetoxification, such as carbonyl reductase (CBR)(EC: 1.1.1.184), aldehyde reductase (EC: 1.1.1.2)and dihydrodiol dehydrogenase (EC: 1.3.1.20)(Ohara et al., 1995; Propper and Maser, 1997).Carbonyl reductase, whose gene (21q22.12) islocated very close to the SOD-1 locus (21q22.11),catalyses the reduction of DNR to the less activity13-hydroxy metabolite, daunorubicinol (DNR-ol).Like SOD-1, this enzyme displays gene-dosageeffect in DS human lymphoblasts at the DNA andmRNA level and can function as a quinone re-ductase with capacity for modulating quinone-mediated oxygen free radicals (Lemieux et al.,1993). DNR-ol formation paralleled an increase inDNR IC50 value in some DNR-resistant cells, andintroduction of a cloned human carbonyl reductasecDNA to K562 leukaemia cells led to a 2–3–foldreduction in DNR toxicity (Ax et al., 2000;Gonzales et al., 1995). Cusack et al. (1993) found

that cardiac dysfunction observed 3–4 days after asingle dose of DNR was not clearly related tooxidative stress, but was associated with the cardiacconcentration of DNR-ol. In various cell types,different mechanisms cooperate in developing ofMDR (multidrug resistance) and these mechanismsdo not necessarily involve elevated CBR activity,e.g. DNR-resistant K562 leukaemia cells, lackingDNR-reductase activity, displayed 22- to 123-foldresistance to DNR and were cross-resistant to avariety of drugs (Ahmed and Vasanthakumar,1987).

In contrast, several oncological pediatric groups(Kaspers et al., 1998; Taub et al., 1996, 1997, 1999)recently demonstrated that ALL and ANLL cellsfrom DS patients showed increased sensitivityto ara-C, anthracyclines and mitoxantrone. Taubet al. (1999) hypothesised that enhanced sensitivityof DS myeloblasts reflects the altered expressionof chromosome 21-localised genes in DS cellsand suggested that at least two such genes,SOD-1 and cystathionine �-synthase (EC: 4.2.1.22)(CBS; 21q22.3) may directly or indirectly con-tribute to the increased sensitivity to ara-C and


0.00

0.35


12-A

S f

luor

esce

nce

an

isot

ropy

7550201052

0.30

0.25

0.20

0.15

0.10

0.05

0 100

**

*

*

* *

Fig. 6. Effect of DNR on fluorescence anisotropy of 12-AS in normal (S-126, ) and trisomic (S-240, �; and T-164, ) humanfibroblasts. Data are expressed as mean�standard deviation for eight independent experiments. *Significantly different whencompared to values obtained for the S-126 cell line (P<0.001).

daunorubicin. Although levels of CBS and SOD-1mRNA transcripts found in all DS AML sampleswere significantly higher than in non-DS samples,no significant correlation between SOD-1 tran-script level and sensitivity to ara-C and DNR wasdetected, indicating that SOD-1 was not directlyinvolved in the metabolism of either ara-C orDNR. It has been suggested that increased free-radical generation has an additive effect throughlipid peroxidation, damage to cell membrane andother organelles (Hu et al., 1995). Since no signifi-cant differences between the DS and non-DSsamples of AML patients in transcript levels forother chromosome 21-localised genes, includingcarbonyl reductase were found, one might specu-late that, despite the gene dosage effect, theseenzymes are expressed differently in particular celltypes (Taub et al., 1999).

Both DS fibroblast cell lines, investigated in thiswork, like those used by Rozga et al. (1990, 1994),display increased levels of SOD-1 (about 50%) andGSH-Px (about 30%), and similar catalase activity

compared to non-DS fibroblasts (data not shown).Our results indicated decreased sensitivity of DSfibroblasts to DNR (P<0.01) (Figs 1 and 2), whichis in accord with their results showing lower sensi-tivity to carminomycin in five different DS fibro-blast cell lines. Taken together these findingssuggest that, at least in these DS fibroblast celllines, cell damage by DNR and carminomycinanthracyclines is mediated by the superoxide anionand/or reactive oxygen species produced by it.Since leukaemic cells, among others, display manyoncogene mutations and chromosomal aberrationsleading to general genomic instability (Bunin et al.,1991; Mitelman et al., 1991; Haglund et al., 1997;Finette et al., 1998; de Souza Fernandez et al.,2000), these mutations are present additionally tothe 21 trisomy in myeloblasts/lymphoblasts fromDS patients. Thus, it cannot be excluded thatobserved greater sensitivity of DS myeloblasts/lymphoblasts to some anticancer drugs comparedto non-DS fibroblasts might also result from thegeneral genomic instability of leukaemic DS cells.


Efficacy of DNR depends on its concentrationin the target compartments of the cell. Marieet al. (1993) showed that intracellular DNRaccumulation correlated well with the acutemyeloid leukaemia patient outcome. From thispoint of view, the mechanism of transport into thecell and, in particular, the kinetics of transport arecrucially important. Since the uptake of drugs suchas anthracyclines occurs by passive diffusion of itsneutral form (Frezard and Garnier-Suillerot, 1998),nevertheless the results obtained by Nagasawaet al. (1996) indicated that DNR might also involvea carrier-mediated transport, but the mechanism isunknown.

We have compared the dynamic of DNR trans-port through cellular membranes of normal andtrisomic cells. Measurement of the DNR fluor-escence emission intensity demonstrated that DNRwas rapidly taken up by fibroblasts. The steady-state of the intracellular distribution of DNR, infibroblasts growing as a monolayer was reachedwithin 40 min in trisomic fibroblasts, and took60 min in normal cells (Fig. 2). The comparison ofthe kinetic parameters, derived from curves repre-senting the time-dependence of amount of drugtaken up and amount of drug excluded by cells(Table 1, Fig. 2), indicated that the amount of drugtaken up by cells, within 60 min of incubation, wasconsiderably greater for normal than for trisomicfibroblasts. Contrary to this, no statistically signifi-cant difference in the amount of DNR removed bynormal and trisomic cells was observed. These dataare in agreement with results of Koter et al. (1982),who found that the transport of hydrophobic spinlabel probe, TEMPO, across the erythrocyte mem-brane, studied by ESR, was significantly slower forDown’s syndrome patients than for normal donors.

Analysis of intracellular distribution of DNR(Fig. 3) suggests that only a small amount ofcompound incorporated into cells was associateddirectly with DNA. At the same time, almost equalamounts of drug were bound to DNA in bothnormal and trisomic cells. Therefore, part of DNRmolecules, considered free might be accumulated inother cell structures than nucleus. The observeddifferences in the effect of DNR on the survival ofnormal and trisomic cells (Figs 1 and 2) mightpartly be related to the interaction of drug withplasma membrane, another important target forDNR cytotoxic activity.

We used the spectrofluorimetric method tomeasure the fluorescence anisotropy of the lipidprobes TMA-DPH and 12-AS as an assay ofmembrane fluidity, its major advantage being thatthese dyes are confined primarily to the plasma

membrane (Kuhry et al., 1985). Comparison ofanisotropy values obtained by using these fluor-escent probes (Figs 5 and 6) indicated that theuntreated trisomic fibroblasts had lower fluidity(P<0.01) at the surface of the lipid bilayer thanuntreated normal cells, which suggests greaterorder in membrane of trisomic cells. Kantar et al.(1992) demonstrated that erythrocytes derivedfrom DS individuals exhibited lowered cell mem-brane fluidity at the lipid–water interface region,and that lower fluidity at the outer surface lipidpolar heads was unrelated to membrane lipid com-position. Differences in membrane fluidity may beconnected to the changes in membrane fatty acidunsaturation and lipid peroxidation mediated byreactive oxygen species (Pastor et al., 1998).

We indicated (Fig. 5) that, in both cell types,DNR exerted no statistically significant effect onthe membrane fluidity at the outer surface lipidpolar heads, even at the highest doses, but therewas a statistically significant increase in the rigidityof the hydrophobic core of the membrane (Fig. 6),especially at high drug concentrations, suggestingthat the difference in DNR interaction with plasmamembranes of normal and trisomic fibroblasts isnot related to the membrane lipid composition,and confirming the data of Frezard and Garnier-Suillerot (1998). These authors found that theanionic lipid content of the model membrane hadno effect on the global embedding of anthracyclinedrugs, which depends solely on the lipophilicity ofthe drug. Goldman et al. (1978) indicated that thepartitioning of anthracycline aminoglycosides intocell membrane was determined by the membranelipid fluidity and it was assumed as the criticalfactor in the potency of anthracycline drugs. Celluptake of drug probably also depends on its associ-ation with membrane proteins that determines thefree drug gradient between extra- and intracellularspaces. According to Prosperi et al. (1985), DNRsignificantly affects membrane permeability proper-ties and its influence is similar to that exerted bymetabolic inhibitors.

A substantial part of our work has been focusedon the interaction of DNR with plasma membrane,and compared the dynamics of DNR incorporationinto the membranes of normal and trisomic cells.The measurement of fluorescence resonance energytransfer from fluorescent probe TMA-DPH toDNR molecules demonstrated that DNR was rap-idly taken up and distributed in the cell membrane.The steady state of TMA-DPH fluorescencequenching by DNR molecules in the outer leaflet ofthe cell membrane lipid bilayer was reached withinthe first minute of incubation in both normal and


trisomic fibroblasts with DNR (Fig. 4). Compari-son of the diminution in the TMA-DPH fluor-escence emission intensity indicated that the rapidinitial quenching, as well as the second-phasequenching, were lower for both trisomic fibroblastlines than normal cells. Changes in membranefluidity induced by DNR and differences in drugincorporation into cellular membranes suggest thatthe plasma membrane is an important place ofDNR accumulation and action, and may be crucialfor its cytotoxicity.

Anthracycline cytotoxicity might be directly re-lated to plasma membrane composition (Schreier,1989). Daunorubicin was shown to bind, withhigh-affinity constant, to negatively-charged phos-pholipids, such as cardiolipin and phosphatidyl-serine. Cell membranes derived from DS individ-uals showed no significant differences in choles-terol, phospholipid phosphorus content andcholesterol/phospholipid molar ratio (Kantar et al.,1992), suggesting that difference in the DNR cyto-toxic action in normal and trisomic fibroblasts isnot directly related to membrane lipid composition.

In conclusion, the data presented in this paperdemonstrate that lower membrane fluidity ob-served in trisomic fibroblasts imply a lower par-tition coefficient of DNR in the cell membrane,diminished influx rate and decreased drug accumu-lation level. Finally, lower partition coefficient ofDNR and reduced intracellular accumulationin trisomic cells could be responsible for theirincreased survival.

ACKNOWLEDGEMENTS

Authors wish to thank Joanna Lipecka, AnnaBoguslawska and Sylwia Dragojew for their excel-lent technical assistance and Krzysztof Ilnicki forstorage of the fibroblast lines in Tissue Bank of theCentre of Child Health (Warsaw, Poland).

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