CD26/dipeptidyl peptidase IV enhances expression of topoisomerase II alpha and sensitivity to apoptosis induced by topoisomerase II inhibitors

Abstract

CD26/dipeptidyl peptidase IV (DPPIV) is a cell surface-bound ectopeptidase with important roles in T-cell activation and tumour biology. We now report that CD26/DPPIV enhances sensitivity to apoptosis induced by the antineoplastic agents doxorubicin and etoposide. In particular, CD26/DPPIV presence is associated with increased susceptibility to the mitochondrial pathway of apoptosis, documented by enhanced cleavage of poly (ADP ribose) polymerase (PARP), caspase-3 and caspase-9, Bcl-xl, and Apaf-1, as well as increased expression of death receptor 5 (DR5). We also show that the caspase-9-specific inhibitor z-LEHD-fmk inhibits drug-mediated apoptosis, leading to decreased PARP and caspase-3 cleavage, and reduced DR5 expression. Importantly, through detailed studies that demonstrate the association between topoisomerase II alpha expression and DPPIV activity, our data provide further evidence of the key role played by CD26 in biological processes.

Figure 1

Enhancing effect of CD26/DPPIV surface expression on apoptosis induced by topoisomerase II inhibitors. CD26 Jurkat transfectants were incubated at 37°C in culture media alone or culture media containing etoposide (A) for 14 h or doxorubicin (B) for 16 h at the concentrations indicated. Cells were then harvested and Annexin V/PI assays were performed as described in Materials and Methods. wtCD26: wild-type CD26 Jurkat transfectant; S630A: Jurkat cells transfected with mutant CD26 containing an alanine at the putative catalytic serine residue at position 630, resulting in a mutant CD26-positive/DPPIV-negative Jurkat transfectant; control: nontransfected parental Jurkat; 340–4: Jurkat cells transfected with mutant CD26 containing point mutations at the ADA-binding site residues 340–343, with amino acids L₃₄₀, V₃₄₁, A₃₄₂, and R₃₄₃ being replaced by amino acids P₃₄₀, S₃₄₁, E₃₄₂, and Q₃₄₃, resulting in a mutant CD26-positive/DPPIV-positive mutant CD26 Jurkat transfectant incapable of binding ADA. Data are representative of three separate experiments. (C) wtCD26 Jurkat transfectants and parental cells were treated with doxorubicin over the indicated time intervals and drug concentrations. a: 12 h, b: 24 h, c: 36 h. Data are representative of three separate experiments.

Figure 1

Figure 2

CD26/DPPIV-associated enhancement in PARP cleavage induced by topoisomerase II inhibitors. CD26 Jurkat transfectants were incubated at 37°C with media containing etoposide for 16 h or doxorubicin for 18 h at the indicated doses. Cells were then harvested, and whole-cell lysates were obtained. Following SDS–PAGE of lysates, immunoblotting studies for PARP and β-actin were performed as described in Materials and Methods. The cleaved product of PARP was detected at ∼85 kDa. Each lane was loaded with 30 μg of protein.

Figure 3

Time course study of the effect of CD26/DPPIV surface expression on etoposide-induced apoptosis. Jurkat cells were incubated at 37°C with media containing 3 μM etoposide or 1 μM doxorubicin for the indicated time periods at the indicated doses. Cells were then harvested, and cytosol fractions were obtained as described in Materials and Methods. Following SDS–PAGE of lysates, immunoblotting studies with specific antibodies for PARP, caspase-9, caspase-3, Apaf-1, Bcl-xl, and β-actin were performed as described in Materials and methods (^*): caspase-3 cleaved products; (^**): Bcl-xl cleaved products. Each lane was loaded with 30 μg of protein.

Figure 4

Effect of caspase-9 inhibitor z-LEHD-fmk on etoposide-induced apoptosis in wtCD26 Jurkat transfectant. wtCD26 Jurkat transfectants were incubated at 37°C for 2 h of preincubation with z-LEHD-fmk at varying doses, and then treated with 3 μM etoposide or 1 μM doxorubicin for 16 h. Cells were then harvested, and whole-cell lysates were obtained as described in Materials and Methods. Following SDS–PAGE of lysates, immunoblotting studies for PARP, caspase-3, caspase-9, and β-actin were performed as described in Materials and Methods (^*): caspase-3 cleaved products. Each lane was loaded with 30 μg of protein.

Figure 5

Effect of inhibition of DPPIV activity on topoisomerase II alpha expression. (A) After incubation of Jurkat cells at 37°C for 24 h in culture media, cells were harvested and nuclear extracts were obtained. Following SDS–PAGE of lysates, immunoblotting studies were performed for topoisomerase II alpha or β-actin as described in Materials and Methods. Each lane was loaded with 30 μg of protein. Lane 1: wtCD26 Jurkat transfectant, lane 2: S630A mutant transfectant, lane 3: parental Jurkat. (B) wtCD26 Jurkat transfectants or parental Jurkat were incubated in culture media alone (DFP−), culture media containing 100 μM DFP for 2 or 6 h (DFP+). A representative sample of cells reflecting each treatment condition was obtained, and DPPIV enzyme activity assays were then performed as described in Materials and Methods. (C) wtCD26 Jurkat transfectants (lanes 1–3) or parental Jurkat (lanes 4–6) were incubated in culture media alone (lanes 1, 3), culture media containing 100 μM DFP for 2 h (lanes 2, 5) or for 6 h (lanes 3, 6). Cells were harvested, and nuclear extracts were obtained. Following SDS–PAGE of lysates, immunoblotting studies for topoisomerase II alpha or β-actin were performed as described in Materials and Methods. Each lane was loaded with 30 μg of protein. (D) wtCD26 Jurkat transfectants were incubated in culture media (bar I), or in culture media with 100 μM DFP for 4 h (bar II), or they were incubated in culture media with 100 μM DFP for 4 h, then washed twice in PBS to ensure removal of DFP followed by incubation in culture media for 2 h (bar III) or 8 h (bar IV). A representative sample of cells reflecting each treatment condition was obtained, and DPPIV enzyme activity assays were then performed as described in Materials and Methods. (E) wtCD26 Jurkat transfectants were incubated in culture media (lane 1), or in culture media with 100 μM DFP for 4 h (lane 2), or they were incubated in culture media with 100 μM DFP for 4 h, then washed twice in PBS to ensure removal of DFP followed by incubation in culture media for 2 h (lane 3) or 8 h (lane 4). Cells were then harvested and nuclear extracts were obtained. Following SDS–PAGE of lysates, immunoblotting studies for topoisomerase II alpha or β-actin were performed as described in Materials and Methods. Each lane was loaded with 30 μg of protein.

Figure 6

Effect of soluble CD26 molecules on topoisomerase II alpha expression. Parental Jurkat cells (A) or Jiyoye cells (B) were incubated overnight in culture media alone (−) or culture media containing soluble CD26 (sCD26) molecules (300 μg ml⁻¹) (+) at 37°C. Cells were then harvested and nuclear extracts were obtained. Following SDS–PAGE of lysates, immunoblotting studies for topoisomerase II alpha or β-actin were performed as described in Materials and Methods. Each lane was loaded with 30 μg of protein.

Figure 7

CD26-associated enhancement of doxorubicin or etoposide-induced PARP cleavage. Parental Jurkat cells were incubated overnight in culture media alone (−) or culture media containing soluble CD26 (sCD26) molecules (300 μg ml⁻¹) (+) at 37°C, followed by incubation with doxorubicin or etoposide at the indicated concentrations for 16 h. Cells were then harvested, and whole-cell lysates were obtained as described in Materials and Methods. Following SDS–PAGE of lysates, immunoblotting studies for PARP or β-actin were performed as described in Materials and Methods. Each lane was loaded with 30 μg of protein.

Figure 8

Effect of CD26/DPPIV on DR5 expression induced by etoposide treatment. (A) Jurkat cells were incubated at 37°C in culture media containing etoposide (3 μM) for the indicated time periods at the indicated doses. Cells were then harvested, and whole-cell lysates were obtained as described in Materials and Methods. Following SDS–PAGE of lysates, immunoblotting studies for DR5 and β-actin were performed as described in Materials and Methods. Each lane was loaded with 30 μg of protein. Anti- DR5 mAb detects two bands of 58 and 32 kDa. (B) Following 2 h of preincubation at 37°C with varying doses of z-LEHD-fmk, wtCD26 Jurkat transfectants were treated with 3 μM etoposide or 1 μM doxorubicin for 48 h. Cells were then harvested, and whole cell lysates were obtained as described in Materials and Methods. Following SDS–PAGE of lysates, immunoblotting studies for DR5, caspase-9, and β-actin were performed as described in Materials and Methods. Each lane was loaded with 30 μg of protein.