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Dipropylcyclopentylxanthine Triggers Apoptosis in Jurkat T Cells by a Receptor-Independent Mechanism

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Dipropylcyclopentylxanthine Triggers Apoptosis in Jurkat T Cells by a Receptor-Independent Mechanism Cell Death and Differentiation (1997) 4, 639 ± 646 1997 Stockton Press All rights reserved 13509047/97 $12.00 Dipropylcyclopentylxanthine triggers apoptosis in Jurkat T cells by a receptor-independent mechanism 1 1 1 2+ Maribel Mirabet , Josefa Mallol , Carmen Lluis and Rafael apoptosis follows an increase in [Ca ]i, requires protein Franco1,2 synthesis, and is manifested by endogenous nuclease activation which results in DNA fragmentation. At the optical 1 Departament de BioquõÂmica i Biologia Molecular, Facultat de QuõÂmica, microscopy level, apoptosis can be suspected in cells Universitat de Barcelona, Catalonia, Spain presenting a nucleus with condensed chromatin (reviewed 2 correspondence author: Rafael Franco, Departament de BioquõÂmica i Biologia in McConkey et al, 1990; Cohen, 1993). Molecular, Facultat de QuõÂmica, MartõÂ FranqueÁs 1, 08028 Barcelona, Spain. Purine compounds have been widely used in the therapy tel: 343-4021208; fax: 343-4021219; email: [email protected] of proliferative diseases. 6-mercaptopurine and 6-thiogua- Received 24.3.97; accepted 12.6.97 nine were among the first to be used (Hitchings, 1991). Edited by M.L. Gougeon These compounds are metobolized in the cell and some of the resulting products interfere with DNA synthesis. Thus, replication of DNA in cancer cells does not occur and tumor Abstract cell proliferation is blocked. Recent studies have demon- strated that several purine analogs used as anticancer drugs 1,3-Dipropyl-8-cyclopentylxanthine (DPCPX), a xanthine are cytotoxic because they induce apoptosis in proliferating analog used as selective antagonist of adenosine receptors, cells. Thus, 2-chloro-2'-deoxyadenosine and 9-b-D-arabino- caused apoptosis in a human leukemia T cell line. Jurkat cells syl-2-fluoroadenine are potent inducers of apoptotic death in treated with DPCPX underwent apoptosis as demonstrated by lymphocytes from patients with chronic lymphocytic leuke- flow cytometry, by DNA fragmentation and by accumulation of mia (Carrera et al, 1991; Robertson et al,1993).The histones, H2A, H2B, H3 and H4, in the nucleoplasm of cells. molecular mechanism of death induction is not known but Cell cycle and cell sorting analyses indicated an arrest of cells Hentosh and Grippo (1994) have presented evidence that 2- chloroadenine incorporated into DNA may inhibit daughter inG /Mfollowed bythe appearanceof apoptotic cells in G and 2 1 strand synthesis thus contributing to the cytotoxic effects of G /M phases. The mechanism of programmed cell death does 2 2-chloro-2'-deoxyadenosine and its triphosphate derivative not seem to be mediated by signal transduction events at the (cladribine), which is also used as an antileukemic agent. plasma membrane since it did not involve activation of cell The purine nucleoside adenosine is able to cause membrane receptors and modification of the intracellular apoptosis in human thymocytes by a mechanism that levels of Ca2+ or cAMP. Apoptosis by incorporation into DNA involves interaction with specific adenosine receptors of a derivative of DPCPX is suggested in basis of the presence located on the cell surface and which are coupled to of radioactivity label in the DNA obtained from cells heterotrimeric G proteins. Agonists to these receptors preincubated with [3H]DPCPX. produce variations in the intracellular level of second messengers, cAMP and Ca2+, that seem responsible for Keywords: apoptosis; 1,3-Dipropyl-8-cyclopentylxanthine; T the activation of the apoptotic events (Kizaki et al,1990; lymphocytes; leukemia; therapy Szondy, 1994). In contrast to these findings in non malignant human T cells, adenosine triggers apoptosis in human leukemia HL-60 cells by a receptor-independent Abbreviations: BSA, bovine serum albumin; DPCPX, 1,3- Dipropyl-8-cyclopentylxanthine mechanism that requires the entry of the nucleoside to the cell (Tanaka et al, 1994). In this paper we present evidence that a xanthine derivative, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), activates the apoptotic machinery in Jurkat cells, which Introduction derive from a human acute T leukemia. Apoptosis (programmed cell death) and necrosis are the two mechanisms by which nucleated eukaryotic cells die (Duvall and Wyllie, 1986). Necrosis is considered as a pathological Results event in response to major perturbations in the cellular environment. Necrotic cells swell leading to rupture of plasma DPCPX-induced apoptosis in Jurkat cells and organelle membranes, release of cell content and loss of The xanthine derivative, 1,3-dipropyl-8-cyclopentylxanthine organized structure (Eastman et al, 1994). On the other hand, (DPCPX), is used as selective antagonist of A1 and A2b apoptosis is considered as a physiological process taking part adenosine receptors present on the surface of cells. In T in homeostatic regulation; in this way death is necessary in lymphocytes, the most abundant adenosine receptor is the normal tissue turnover (Duvall and Wyllie, 1986; McConkey et A2b subtype, which mediates increases in cAMP, further al, 1989; Rothenberg, 1990). The mechanism(s) of apoptosis potentiated by activation via the receptor for the antigen may differ from one system to another. In most cases, (Fredholm and Sandberg, 1983; Kvanta and Fredholm, 1991). Apoptosis by dipropylcyclopentylxanthine in Jurkat T lymphocytes MMirabetet al 640 We were interested in determining whether agonists or independent since they were not antagonized by N- antagonists of adenosine receptors modulate T cell activa- ethylcarboxamido adenosine. tion. We found that N-ethylcarboxamido adenosine (up to The presence of apoptotic bodies in cells treated for 250 mM, 24 ± 72 h period), an agonist of A2b receptors, did not 48 h with 25 mM DPCPX led to the suspicion that the modify the proliferative capacity of Jurkat cells whereas reagent initiated a death cell program in Jurkat T cells DPCPX had an antiproliferative effect. In fact, concentrations (Figure 1). The laser-beam flow cytometric analysis of of 10 mM DPCPX or higher led to a marked decrease in the untreated cells indicated a single population whereas number of cells and in the [3H]thymidine incorporation. The treated cells presented two populations, one similar to percentage of reduction in the number of cells in chronic that of untreated cells and the other displaying decreased treatments (up to 144 h) with 25 mM DPCPX approached forward light scattering (FSC) and increased side light 90%. This was not due to a slower rate of proliferation but to scattering (SSC) (Figure 1). The combination of high side cytotoxicity since it was accompanied by a reduction of the scattering and low forward scattering is an indication of viability of the cells. These effects of DPCPX were receptor- apoptotic cells (Darzynkiewicz et al, 1992; Dive et al, 1992). Figure 1 Morphology of DPCPX-treated Jurkat cells. The forward (cell size) versus side scatter (properties of cytoplasmic and nuclear components) distribution of naõÈve Jurkat cells (upper part) or cells treated with 25 mM DPCPX for 48 h (lower part). The percentage of cells in each population respect to the total amount of cells analyzed by flow cytometry is given. Inserts: micrographs representing the total population (3006) Apoptosis by dipropylcyclopentylxanthine in Jurkat T lymphocytes MMirabetet al 641 To confirm the presence of DPCPX-induced apoptosis in highly permeable plasma membranes. The presence of Jurkat cells, a set of complementary experiments was cells with a pycnotic nucleus but with an intact plasma carried out. First, the effect of DPCPX upon Jurkat's cell membrane, i.e. not labeled with propidium iodide, is an cycle was analyzed. For this purpose the DNA of the cells indication that death comes as a consequence of (untreated or treated) was labeled with Hoechst 33342 (see apoptosis. Analysis of fluorescence at 395 nm and Materials and Methods). In control cells the pattern did not 675 nm in treated cells revealed B1, B2, B1' and B2' change significantly over time; the percentage of cells in populations (See Figure 3A bottom); two were similar to G0/G1 phase was 37 ± 44 whereas the percentage in S those found in control cells (B1 and B2) whereas the other phase was 41 ± 44. Treatment of cells with DPCPX led to a two (B1' and B2') emitted more fluorescence at 675 nm. A progressive accumulation of fluorescence near the Y-axis fifth population corresponding to small DNA fragments in due to DNA fragments released from dead cells. After 8 h apoptotic bodies or released to the culture medium was of treatment, DPCPX led to the accumulation of cells in the located near the axis origin and was not further analyzed. A G2/M phase (Figure 2). At a longer time (24 h) a large cell sorter permitted the isolation of the four different proportion of DPCPX-treated cells was found in a peak populations, which were then observed under an epifluor- previous to that denoting the G1 phase (Figure 2). This escence microscope. All cells among B1 and B2 peak, which is characteristic of apoptotic cells, corresponds populations had a normal appearance. In contrast, all to cells with fragmented DNA (Gorzyca et al, 1993; Belloc cells of B1' and B2' populations were apoptotic, as et al, 1994; Zhu and Anasetti, 1995). At 48 h of treatment, indicated by the pycnotic nucleus (Figure 3B). Thus, the peak corresponding to cells with fragmented DNA was DPCPX produced apoptotic B1' cells that probably derive also present. A fourth peak prior to that representing the from cells initially in the G1 phase and apoptotic B2' cells G2/M phase appeared after 24 or 48 h of treatment. Thus, that probably derive from cells initially in G2/M phase. The the xanthine analog induced the appearance of a double number of cells having a pycnotic nucleus increased with cell cycle with the two standard peaks representing G1 and DPCPX treatment in a dose-dependent fashion and was G2/M phases and two extra peaks. It should be noted that evident already at 5 mM (at 24 or 48 h of treatment).
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