Tài liệu Báo cáo khoa học: A comparative analysis of the time-dependent antiproliferative effects of

A comparative analysis of the time-dependent antiproliferative

effects of daunorubicin and WP631

Silvia Villamarı´n1,*, Sylvia Mansilla1,*, Neus Ferrer-Miralles1

, Waldemar Priebe2 and Jose´ Portugal1

l

Departamento de Biologı´a Molecular y Celular, Instituto de Biologı´a Molecular de Barcelona, CSIC, Barcelona, Spain; 2

Department of Bioimmunotherapy, The University of Texas M. D. Anderson Cancer Center, Houston, TX, USA

Jurkat T lymphocytes were treated with daunorubicin and

WP631, a daunorubicin-based DNAbinding agent, in

experiments aimed to analyze cellular uptake of these drugs

and their effect on cell viability. WP631 was taken up more

slowly than daunorubicin, but laser confocal microscopy

and spectrofluorometric quantification showed that the drug

accumulated in the cells. Despite the slow uptake rate, the

antiproliferative capacity of WP631 (measured as IC50 after

a 72-h continuous treatment) was greater than that of

daunorubicin. The propensities of daunorubicin and WP631

to promote apoptosis were compared. Our results indicate

that the major effect of WP631 was a G2/M arrest followed,

after about 72 h of treatment, by polyploidy and mitotic

(reproductive) death. In contrast, daunorubicin induced a

rapid response with classic features of apoptosis.

Keywords: anthracyclines; p53; cell-cycle; mitotic catastro￾phe; Jurkat T lymphocytes.

Anthracyclines are among the most potent and clinically

useful drugs in cancer treatment [1]. Anthracycline anti￾biotics are DNAintercalators [2,3], and the antitumor

activity of daunorubicin, a prominent member of this group

of antibiotics, may be associated with its binding to DNA,

although several mechanisms have been proposed to fully

explain the cytotoxic actions of these antitumor molecules

[1,4,5].

Detailed information on the structural and thermo￾dynamic basis of daunorubicin binding to DNA[2,3,6] has

provided the foundation upon which to design WP631, a

new bisanthracycline (Fig. 1) resulting from a
Modular

Design Approach [7]. WP631 bisintercalates into DNA,

and displays enhanced binding affinity and sequence

selectivity over monomeric daunorubicin [8]. These charac￾teristics make WP631 a more effective antitumor drug

against some cell lines, including a multidrug-resistant one

[8,9]. Moreover, there are grounds for considering that

WP631 is a potent inhibitor of transcription through direct

competition with transcription factors [9–11].

Anthracyclines induce apoptosis, although this might be

the final cell response to other events such as unpairing of

DNAreplication or inhibition of transcription and topo￾isomerase activity [4,12]. Interaction of anthracyclines with

DNA-topoisomerase II complexes may trigger apoptosis. In

Jurkat T lymphocytes, daunorubicin, and the related drug

doxorubicin, are considered inductors of apoptosis [13].

Nevertheless, this effect may only be true for some cell types

or drugs, as the onset of apoptosis appears to depend on the

cell line [4,14]. Alternatively, G2 arrest by anthracyclines may

result from the disruption of some cell cycle activities [15,16],

and thus in some cases the rapid induction of apoptosis may

not be the main mechanism leading to cell death [17].

Despite the potent effect of WP631 on the viability of

Jurkat cells [9], continuous treatment over 72 h produces

only marginal apoptosis. Arrest in G2 after treatment, which

depends on the levels of p53 protein [16], suggests that the

extent of p53-dependent apoptosis is not a critical factor in

the sensitivity to WP631 [16]. Although it is widely accepted

that the sensitivity of cells to damaging agents, including

anthracyclines, might reflect cell death by apoptosis [18], the

relationship between the efficacy of drug treatment and the

induction of apoptosis is still an open issue [19,20]. Here we

show that low concentrations of WP631 produce nonapop￾totic cell death, in contrast with monomeric anthracyclines

that can produce nonapoptotic tumor cell death only at high

(supraclinical) concentrations [4]. Genomic site damage may

explain the differences in drug efficacy between the mono￾intercalating anthracyclines and the more sequence-selective

bisanthracycline WP631. To gain further insight into the

causes of the distinct behavior of daunorubicin and WP631,

we compared the intracellular accumulation of these com￾pounds in Jurkat T cells overtime. We also examined the

rate and overall level of cell killing by either drug by

apoptosis or mitotic, reproductive, death after G2 arrest.

Materials and methods

Daunorubicin and WP631

Solutions containing 500 lM daunorubicin (Sigma) or

WP631 were prepared with sterile 150 mM NaCl, main￾tained at )20
C, and brought to the final concentration

with RPMI 1640 medium just before use.

Correspondence to J. Portugal, Departamento de Biologı´a Molecular

y Celular, Instituto de Biologı´a Molecular de Barcelona, CSIC.,

Jordi Girona, 18–26, 08034 Barcelona, Spain.

Fax: + 34 93 204 59 04, Tel.: + 34 93 400 61 76,

E-mail: [email protected]

Abbreviations: MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetra￾zolium bromide.

*Note: these authors contributed equally to this work.

(Received 29 August 2002, revised 29 October 2002,

accepted 19 December 2002)

Eur. J. Biochem. 270, 764–770 (2003) FEBS 2003 doi:10.1046/j.1432-1033.2003.03442.x