Apoptosis induces Bcl-XS and cleaved Bcl-XL in chronic lymphocytic leukaemia

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Abstract

The Bcl-X gene has both pro-survival, Bcl-XL, and pro-apoptotic, Bcl-XS, gene products, which are produced by alternative splicing. The function of these proteins has previously been characterised in cell lines, often by transfecting expression constructs, and primary cell systems capable of dynamically regulating Bcl-XL and Bcl-XS have not been described. Such a system is potentially important to allow testing of agents that promote apoptosis by increasing the amount of Bcl-XS at the expense of Bcl-XL. In this report we characterise Bcl-X gene products in primary human leukaemic B-cells in culture conditions associated with survival and apoptosis. We found that Bcl-XS was induced in spontaneous and drug-induced apoptosis and that apoptosis induced in cells cultured on mouse fibroblasts expressing CD40 ligand with IL-4 (CD154/IL-4), a condition mimicking the tissue microenvironment, additionally produced expression of cleavage products of Bcl-XL. Both Bcl-XS and Bcl-XL were produced in a caspase dependent manner. We tested emetine, an agent previously reported to increase Bcl-XS but found that it did not have this effect in primary human B-cells. Therefore, there are two mechanisms—cleavage of Bcl-XL and production of Bcl-XS—by which Bcl-X gene products could enhance apoptosis in CLL but neither appeared to have a primary role in inducing leukaemic cell death.

Research highlights

► Bcl-X gene has both pro-survival, Bcl-XL, and pro-apoptotic, Bcl-XS, gene products. ► No well-characterised primary cell systems to test drugs operating through this mechanism exist. ► Bcl-XS and cleaved Bcl-XL were induced in a caspase dependent manner. ► Cleavage of Bcl-XL and production of Bcl-XS both enhance apoptosis. ► Emetine induced apoptosis does not depend on Bcl-XS in primary human leukaemic B-cells.

Introduction

Chronic lymphocytic leukaemia is a common lymphoproliferative disorder in which there is an accumulation of mature lymphocytes in the peripheral blood, lymph nodes and bone marrow. Diverse treatments, using both conventional and novel therapeutic agents, clear circulating leukaemic cells but leukaemic cells continue to survive within the lymph node and bone marrow microenvironments. A variety of model systems [1], [2], [3] have been developed to attempt to reproduce the humoral or cellular aspects of the microenvironment responsible for their improved survival. We are using CD40 ligand (CD154) expressed on the surface of a mouse fibroblast cell line with IL-4 [4], [5], [6] and have found that this causes increased proliferation and resistance to drug-induced and spontaneous apoptosis. Others using the same system have demonstrated that up-regulation of Bcl-2 family pro-survival members Bcl-XL and Bcl2A1 causes improved survival and resistance to apoptosis [7].

Bcl-2 family proteins regulate apoptosis through control of mitochondrial integrity. The Bcl-2 family member, Bcl-X, has two major isoforms Bcl-XL and Bcl-XS, due to alternative splicing [8]. The shorter, Bcl-XS, isoform lacks 63 residues comprising the BH1 and BH2 domains, but has the BH3 and BH4 domains in common with Bcl-XL. Bcl-XL is a pro-survival protein but Bcl-XS is pro-apoptotic [8]. Most work on Bcl-XS expression has been carried out on cell lines.

Utilising cell lines and Bcl-X minigene constructs it has been shown that splicing to the 5′ Bcl-XS site is enforced by a number of RNA binding proteins [9], [10], [11], [12]. More recent work on intracellular signalling pathways has demonstrated that active protein kinase C reduces splicing to Bcl-XS [13].

Ceramides [14] and emetine [15] increase alternative splicing to Bcl-XS in cell lines, suggesting that these agents may form the basis for therapeutically useful strategies. Emetine, a plant alkaloid originally extracted from ipecac root and used as an anti-protozoal agent, increased the ratio of Bcl-XS to Bcl-XL in a C33A cervical cancer cell line [15]. It has also shown potential for use in combination chemotherapy in leukaemias as it seems to increase the efficacy of other drugs such as cisplatin [16]. Ceramide, a cell membrane sphingolipid, was shown to increase Bcl-XS mRNA production at the expense of Bcl-XL [14] in A549 adenocarcinoma cells.

We made the observation that Bcl-XS protein expression is strongly induced during spontaneous apoptosis of CLL cells. Increasing Bcl-XS expression could potentially be exploited therapeutically but it is not known whether Bcl-XS has a primary or secondary role in apoptosis in primary leukaemic B-cells. In this report we analysed the expression of Bcl-X gene products on induction of apoptosis in CLL cells supported on CD154/IL-4.

Section snippets

Cell culture

Blood samples were obtained from patients (Table 1) after informed consent was given. All patients were either untreated or more than 3 months from the end of treatment. Chronic lymphocytic leukaemia (CLL) cells were isolated using density gradient centrifugation. Cells (3 × 106/ml) were cultured in RPMI 1640 medium (Invitrogen, Paisley, UK) supplemented with fetal bovine serum (Invitrogen) and Penicillin/Streptomycin (Invitrogen). Culture conditions were tissue culture plastic alone or co-culture

Bcl-XS protein expression in CLL is induced by spontaneous and drug-induced apoptosis

We investigated Bcl-XL and Bcl-XS expression in primary human leukaemic B-cells. CLL cells survive when cultured on a stromal cell layer (either CD154/IL-4 or NT) but have high rates of spontaneous apoptosis when cultured on tissue culture plastic [5], [17]. By western there is little detectable Bcl-X gene product in freshly isolated CLL cells (Fig. 1A). On culture with CD154/IL-4 Bcl-XL was strongly induced whereas with the fibroblast layer alone (NT) there was a low level of Bcl-XL

Discussion

Signals from the lymph node and bone marrow microenvironments are responsible for the strong survival and proliferation of CLL cells. It is also believed that survival within tissues contributes to the failure of conventional chemotherapy to eliminate leukaemic cells and there is, therefore, interest in developing strategies targeting the lymph node microenvironment and the leukaemic cells existing within this context [1].

We used cell culture conditions to reflect the different

Author contribution

SW designed and carried out experiments. TM carried out experiments. SDW designed experiments and wrote the manuscript.

Conflict of interest

None.

Acknowledgments

ABT-737 was a gift from Abbott Laboratories. This work was supported by a grant from the Kay Kendall Leukaemia Fund to S.D.W.

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