Summary We examined the effectiveness of various anti‐tumour agents to natural killer (NK)‐cell tumour cell lines and samples, which are generally resistant to chemotherapy, using flow cytometric terminal deoxynucleotidyl transferase‐mediated dUTP‐biotin nick end‐labelling (TUNEL) assay. Although NK‐YS and NK‐92 were highly resistant to various anti‐tumour agents, l‐asparaginase induced apoptosis in these two NK‐cell lines. NK‐cell leukaemia/lymphoma and acute lymphoblastic leukaemia (ALL) samples were selectively sensitive to l‐asparaginase and to doxorubicin (DXR) respectively. Samples of chronic NK lymphocytosis, an NK‐cell disorder with an indolent clinical course, were resistant to both drugs. Our study clearly separated two major categories of NK‐cell disorders and ALL according to the sensitivity to DXR and l‐asparaginase. We examined asparagine synthetase levels by real‐time quantitative polymerase chain reaction (RQ‐PCR) and immunostaining in these samples. At least in nasal‐type NK‐cell lymphoma, there was a good correlation among asparagine synthetase expression, in vitro sensitivity and clinical response to l‐asparaginase. In aggressive NK‐cell leukaemia, although asparagine synthetase expression was high at both mRNA and protein levels, l‐asparaginase induced considerable apoptosis. Furthermore, samples of each disease entity occupied a distinct area in two‐dimensional plotting with asparagine synthetase mRNA level (RQ‐PCR) and in vitrol‐asparaginase sensitivity (TUNEL assay). We confirmed rather specific anti‐tumour activity of l‐asparaginase against NK‐cell tumours in vitro, which provides an experimental background to the clinical use of l‐asparaginase for NK‐cell tumours.

ABSTRACT

P-glycoprotein (P-gp), a transmembrane efflux pump encoded by theMDR1 gene, has been found to be expressed in many normal bone marrow and peripheral blood cells. Among normal leukocytes, CD3−CD16+ or CD3−CD56+ lymphocytes, ie, natural killer (NK) cells, express relatively high levels of P-gp, but little is known about P-gp in abnormally expanded NK cells. In this study, we examined the expression and activity of P-gp on NK cells derived from three normal donors, six patients with indolent NK cell-lineage granular lymphocyte-proliferative disorder (NK-GLPD), three patients with aggressive NK cell tumors (one NK cell leukemia and two nasal NK cell lymphoma), and two NK cell lines. By flow cytometric analysis using the monoclonal antibody (MoAb) MRK16 and rhodamine 123 dye (Rh123), P-gp expression and the efflux of Rh123 were found in all NK samples except one NK cell line. The Rh123 efflux of NK cells was inhibited by cyclosporin A (CsA) and its analogue PSC 833, but the aggressive NK tumor cells were less inhibited than were the other NK cells. The percent inhibition of efflux in the normal NK cells, indolent NK-GLPD cells and aggressive NK cell tumors was 81.8% ± 0.9%, 93.4% ± 3.1% and 36.9% ± 11.7%, respectively, by 1 μmol/L CsA, and 80.2% ± 3.6%, 91.7% ± 2.6% and 32.7% ± 10.1%, respectively, by 1 μmol/L PSC833. In reverse transcription-polymerase chain reaction (RT-PCR) analysis, the low inhibitory effect of P-gp modulators in aggressive NK cell tumors did not correlate to the expression level of MDR1 gene, multidrug resistance-associated protein gene, or human canalicular multispecific organic anion transporter gene. This phenomenon could be related to the presence of other transporters or to unknown cellular or membrane changes. Some patients with NK cell tumors have been reported to show a highly aggressive clinical course and to be refractory to chemotherapy, and this could be related to the expression of P-gp on NK cells. Our results suggest that, although the inhibitors for P-gp have been used in combination with chemotherapy in some hematologic tumors, these inhibitors may be less effective against aggressive NK cell tumors.

Summary. In the normal developmental pathway of natural killer (NK) cells, pre-NK cells express CD161, immature NK cells express CD161 and CD56, and mature NK cells express CD161, CD56 and CD94. To identify the normal counterpart of NK cells from which neoplastic cells originate, surface antigens were analysed. Blastic NKcell lymphoma/leukaemia lacked CD94 and CD161 but had CD56. Aggressive NK-cell leukaemia/lymphoma and nasal NK-cell lymphoma, although morphologically immature, expressed both CD56 and CD94 and strong NK activity. Cells from chronic NK lymphocytosis expressed CD56 and CD94.

Modification of the cytoplasmic tails of the integrinIntegrin ␣ IIb ␤ 3 (GPIIb-IIIa) is one of the receptors on the cellular surface of platelets and megakaryocytes. It binds to various adhesive proteins including fibrinogen, von Willebrand factor, vitronectin, and fibronectin that contain a core amino acid sequence of arginine-glycine-aspartic acids (RGD). Binding of fibrinogen to ␣ IIb ␤ 3 leads to platelet aggregation and finally to thrombus formation at the injured vascular sites. A pivotal role of ␣ IIb ␤ 3 in hemostasis is supported by the clinical observation that the congenital deficiency of ␣ IIb ␤ 3 , Glanzmann's thrombasthenia, results in lifelong bleeding tendency (1). Whereas ␣ IIb ␤ 3 on resting platelets does not bind soluble fibrinogen, once platelets are activated, conformation of the extracellular domains of the ␣ IIb ␤ 3 is altered and its ligandbinding affinity is increased (affinity modulation) (2). This process of the inside-out signaling is considered to be mediated by modification of the short cytoplasmic tails of ␣ IIb and ␤ 3 subunits; however, the mechanism remains to be elucidated.The nuclear magnetic resonance structural analysis of the ␣ IIb cytoplasmic tail revealed a closed conformation where the highly conserved N-terminal membrane-proximal region forms an ␣-helix followed by a turn, and the acidic C-terminal loop interacts with the N-terminal helix (3). Deletion of almost the entire ␣ IIb -cytoplasmic tail and mutations in its N-terminal sequence (GFFKR) conserved among the integrin ␣ subunits enhance the affinity of ␣ IIb ␤ 3 for ligands (4 -6). The cytoplasmic tail of the ␤ 3 subunit also has an amino acid sequence that is conserved among integrin ␤ subunits: a stretch of 8 amino acids (KLLITIHD) adjacent to the transmembrane domain. In a similar fashion to the ␣ IIb subunit, deletion or mutation in this conserved region induces activation of ␣ IIb ␤ 3 (6, 7). These observations suggest that membrane-proximal regions of the cytoplasmic domains of both subunits exert a negative regulatory function and lock ␣ IIb ␤ 3 in a low affinity state. Negative regulation may be mediated by the interaction between ␣ IIb and ␤ 3 cytoplasmic tails, possibly through a salt bridge between Arg-995 in ␣ IIb and Asp-723 in ␤ 3 (6), or binding of intracellular proteins to ␣ IIb and/or ␤ 3 subunits. Two candidates for the modulator proteins have been reported: calcium-and integrinbinding protein (CIB) 1 (8) and ␤ 3 -endonexin (9, 10), which bind to ␣ IIb and ␤ 3 cytoplasmic tails, respectively. Although CIB is unlikely to have a regulatory effect on ␣ IIb ␤ 3 ligand binding function (11), ␤ 3 -endonexin fused to GST protein induces the conformational change of ␣ IIb ␤ 3 and activates it when co-transfected with ␣ IIb and ␤ 3 subunits in Chinese hamster ovary cells. Another mechanism of modification has been recently suggested: an interaction between cytoplasmic tails of ␣ IIb ␤ 3 and the actin cytoskeleton. ␣ IIb ␤ 3 and the actin cytoskeleton are physically linked by binding of talin to the ␤ 3 cy...