Miki Ando

Selective apoptosis of natural killer-cell tumours by L-asparaginase

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.

A Safeguard System for Induced Pluripotent Stem Cell-Derived Rejuvenated T Cell Therapy

Summary The discovery of induced pluripotent stem cells (iPSCs) has created promising new avenues for therapies in regenerative medicine. However, the tumorigenic potential of undifferentiated iPSCs is a major safety concern for clinical translation. To address this issue, we demonstrated the efficacy of suicide gene therapy by introducing inducible caspase-9 (iC9) into iPSCs. Activation of iC9 with a specific chemical inducer of dimerization (CID) initiates a caspase cascade that eliminates iPSCs and tumors originated from iPSCs. We introduced this iC9/CID safeguard system into a previously reported iPSC-derived, rejuvenated cytotoxic T lymphocyte (rejCTL) therapy model and confirmed that we can generate rejCTLs from iPSCs expressing high levels of iC9 without disturbing antigen-specific killing activity. iC9-expressing rejCTLs exert antitumor effects in vivo. The system efficiently and safely induces apoptosis in these rejCTLs. These results unite to suggest that the iC9/CID safeguard system is a promising tool for future iPSC-mediated approaches to clinical therapy.

Hsp90-inhibitor geldanamycin abrogates G2 arrest in p53-negative leukemia cell lines through the depletion of Chk1.

Checkpoint protein Chk1 has been identified as an Hsp90 client. Treatment with 100 nM geldanamycin (GM) for 24 h markedly reduced the Chk1 amount in Jurkat and ML-1 leukemia cell lines. Because Chk1 plays a central role in G2 checkpoint, we added GM to G2-arrested Jurkat and HL-60 cells pretreated with 50 nM doxorubicin for 24 h. GM slowly released both cell lines from doxorubicin-induced G2 arrest into G1 phase. GM also abrogated ICRF-193-induced decatenation G2 checkpoint in Jurkat and HL-60 cells. Western blot analysis showed that addition of GM attenuates doxorubicin- and ICRF-193-induced Chk1 phosphorylation at Ser345. GM, however, failed to abrogate G2 arrest in p53-positive ML-1 cells maybe due to the p21 induction. GM released HeLa cells from doxorubicin-induced G2 arrest but trapped them at M phase. Flow cytometric analysis showed that addition of GM converted doxorubicin-induced necrosis into apoptosis in Jurkat cells. Colony assay indicated that although GM has a weak cytotoxic effect as a single agent, it dramatically intensifies the cytotoxicity of doxorubicin and ICRF-193 in Jurkat and HL-60 cells. These results suggest that abrogation of G2 checkpoint by GM may play a central role in sensitizing p53-negative tumor cells to DNA-damaging and decatenation-inhibiting agents.

Bortezomib sensitizes non-small cell lung cancer to mesenchymal stromal cell-delivered inducible caspase-9-mediated cytotoxicity

Delivery of suicide genes to solid tumors represents a promising tumor therapy strategy. However, slow or limited killing by suicide genes and ineffective targeting of the tumor has reduced effectiveness. We have adapted a suicide system based on an inducible caspase-9 (iC9) protein that is activated using a specific chemical inducer of dimerization (CID) for adenoviral-based delivery to lung tumors via mesenchymal stromal cells (MSCs). Four independent human non-small cell lung cancer (NSCLC) cell lines were transduced with adenovirus encoding iC9, and all underwent apoptosis when iC9 was activated by adding CID. However, there was a large variation in the percentage of cell killing induced by CID across the different lines. The least responsive cell lines were sensitized to apoptosis by combined inhibition of the proteasome using bortezomib. These results were extended to an in vivo model using human NSCLC xenografts. E1A-expressing MSCs replicated Ad.iC9 and delivered the virus to lung tumors in SCID mice. Treatment with CID resulted in some reduction of tumor growth, but addition of bortezomib led to greater reduction of tumor size. The enhanced apoptosis and anti-tumor effect of combining MSC-delivered Ad.iC9, CID and bortezomib appears to be due to increased stabilization of active caspase-3, as proteasomal inhibition increased the levels of cleaved caspase-9 and caspase-3. Knockdown of X-linked inhibitor of apoptosis protein (XIAP), a caspase inhibitor that targets active caspase-3 to the proteasome, also sensitized iC9-transduced cells to CID, suggesting that blocking the proteasome counteracts XIAP to permit apoptosis. Thus, MSC-based delivery of the iC9 suicide gene to human NSCLC effectively targets lung cancer cells for elimination. Combining this therapy with bortezomib, a drug that is otherwise inactive in this disease, further enhances the anti-tumor activity of this strategy.

Fail-Safe System against Potential Tumorigenicity after Transplantation of iPSC Derivatives

Summary Human induced pluripotent stem cells (iPSCs) are promising in regenerative medicine. However, the risks of teratoma formation and the overgrowth of the transplanted cells continue to be major hurdles that must be overcome. Here, we examined the efficacy of the inducible caspase-9 (iCaspase9) gene as a fail-safe against undesired tumorigenic transformation of iPSC-derived somatic cells. We used a lentiviral vector to transduce iCaspase9 into two iPSC lines and assessed its efficacy in vitro and in vivo. In vitro, the iCaspase9 system induced apoptosis in approximately 95% of both iPSCs and iPSC-derived neural stem/progenitor cells (iPSC-NS/PCs). To determine in vivo function, we transplanted iPSC-NS/PCs into the injured spinal cord of NOD/SCID mice. All transplanted cells whose mass effect was hindering motor function recovery were ablated upon transduction of iCaspase9. Our results suggest that the iCaspase9 system may serve as an important countermeasure against post-transplantation adverse events in stem cell transplant therapies.

CD20-positive extranodal NK-cell lymphoma, nasal-type.

To the Editor: A 71-yr-old Japanese man referred to our hospital because of CD20-positive lymphoma of the right thenar. Physical examination showed an ulcerative tumor of 5 cm in the right thenar prominence, and a subcutaneous tumor of 3 cm in the RLQ adjacent to the inguinal region. He had no peripheral lymphadenopathy or hepatosplenomegaly. Laboratory analysis revealed a white blood cell count of 2.7 · 10 ⁄L with 5.5% of large granular lymphocytes, hemoglobin 10.0 g ⁄dL, platelet count 12.2 · 10 ⁄L, T-Bil 2.2 mg ⁄dL, LDH 1, 177 IU ⁄dL, AST 196 IU ⁄L, ALT 159 IU ⁄L and soluble interleukin-2 receptor level of 5340 U ⁄mL. Serological tests were positive for HBs Ag, HCV Ab, and HTLV-1 Ab. The EBV DNA copy number in the peripheral blood was less than 2.0 · 10 copy. A biopsy specimen from the thenar tumor showed diffuse infiltration of mediumsized lymphocytes from the epidermis to subcutaneous tissue (Fig. 1A). Azurophilic granules were evident in the pale cytoplasm of the lymphocytes (Fig. 1B). The cells were positive for CD3, CD20 (Fig. 1C), CD56 (Fig. 1D), TIA1, and granzyme B by immunohistochemical analysis. Flow cytometric analysis confirmed that these cells were positive for CD2, CD20 (Fig. 1E), CD56 (Fig. 1F), and negative for CD3, CD4, CD5, CD7, CD8, CD10, CD16, CD34, j, k, and T cell receptor (TCR)ab. In situ hybridization showed that these cells were positive for EBER. Southern blot analysis detected no rearrangement of immunoglobulin (Ig) heavy chain, TCR-b, c and d chains, but showed monoclonal integration of EBV genome. Monoclonal band of HTLV-1 was absent. The patient was diagnosed with CD20-positive extranodal NK-cell lymphoma, nasal type. Treatment with CHOP, ESHAP, and L-asparaginase failed to induce CR, and the patient died of the disease progression after six months. CD20 is a cell surface antigen widely expressed in various stages of B-cell differentiation and in B-cell lymphomas. Anti-CD20 monoclonal antibody rituximab is very efficient and largely used in the treatment of B-cell malignancies. Not only B cells but also small subsets of mature T lymphocytes in the peripheral blood have been shown to express CD20 (1). To our knowledge, 19 cases of mature T-cell malignancy have been reported to express CD20 (2–6). However, there has been no report of CD20-positive NK-cell tumor. Flow cytometric, immunohistochemical and Southern blot analysis have definitely concluded that the present case is a typical extranodal NK-cell lymphoma, nasal type. Although treatment with rituximab might have improved the clinical course, we failed to treat the patient with rituximab because of the rapid disease progression and delay in

Mesenchymal Stromal Cells for Linked Delivery of Oncolytic and Apoptotic Adenoviruses to Non-small-cell Lung Cancers

Oncolytic adenoviruses (OAdV) represent a promising strategy for cancer therapy. Despite their activity in preclinical models, to date the clinical efficacy remains confined to minor responses after intratumor injection. To overcome these limitations, we developed an alternative approach using the combination of the OAdv ICOVIR15 with a replication incompetent adenoviral vector carrying the suicide gene of inducible Caspase 9 (Ad.iC9), both of which are delivered by mesenchymal stromal cells (MSCs). We hypothesized that coinfection with ICOVIR15 and Ad.iC9 would allow MSCs to replicate both vectors and deliver two distinct types of antitumor therapy to the tumor, amplifying the cytotoxic effects of the two viruses, in a non-small-cell lung cancer (NSCLC) model. We showed that MSCs can replicate and release both vectors, enabling significant transduction of the iC9 gene in tumor cells. In the in vivo model using human NSCLC xenografts, MSCs homed to lung tumors where they released both viruses. The activation of iC9 by the chemical inducer of dimerization (CID) significantly enhanced the antitumor activity of the ICOVIR15, increasing the tumor control and translating into improved overall survival of tumor-bearing mice. These data support the use of this innovative approach for the treatment of NSCLC.

Spindle checkpoint protein Bub1 corrects mitotic aberrancy induced by human T-cell leukemia virus type I Tax

Bub1 is a component of the mitotic spindle checkpoint apparatus. Abnormality of this apparatus is known to cause multinuclei formation, a hallmark of chromosomal instability (CIN). A549, aneuploid cell line, aberrantly passed through the mitotic phase and became multinuclei morphology in the presence of nocodazole. Time-lapse videomicroscopy showed unreported bizarre morphology, which we named ‘mitotic lobulation’ in A549 cells just before the exit from mitosis and multinuclei formation. External expression of wild-type Bub1-EGFP clearly suppressed the multinuclei formation by retaining A549 cells at the mitotic phase during 48 h of time-lapse observation. This suppressive effect on mitotic aberrancy should not be mere restoration of normal Bub1 function, because A549 cells express proper amount of Bub1, which distributed cytoplasm during interphase and concentrated at kinetochore in metaphase. Furthermore, external expression of wild-type Bub1-EGFP suppressed multinuclei formation induced by Tax both in A549 and HeLa cells. Tax is known to induce mitotic abnormality by binding and inactivating Mad1. These observations, therefore, suggest functional redundancy between Bub1 and other mitotic checkpoint protein(s) and a possibility of correction of mitotic aberrancy by external Bub1 expression.

Cytokine‐producing sarcoma mimics eosinophilic leukaemia

To the Editor: In March 2003, a 30-yr-old Japanese woman with a history of malignant fibrous histiocytoma (MFH), the most common type of soft-tissue sarcoma, attended our hospital because of high fever, cough and shortness of breath. She had remarkable eosinophilia of 160 700/ lL, which accounted for 82.4% of total WBC, and immense eosinophilic infiltration into the lungs. Bone marrow examination showed marked proliferation of eosinophils (63.8% of total nucleated cells) and a considerable portion of them were large and morphologically immature (Fig. 1A,B); however cytogenetic analysis revealed a normal karyotype. She had started chemotherapy in October 2001 and had then been operated for MFH in the left knee in March 2002. The peripheral eosinophil count had gradually increased to 3200/lL just before the start of chemotherapy and had decreased to 100/lL after the surgical resection of the tumour. When much severe eosinophilia was noticed in March 2003, however, orthopaedic examination ruled out the recurrence of MFH. Because of remarkable proliferation of immature eosinophils, she was diagnosed to have idiopathic hypereosinophilic syndrome probably because of the autonomous proliferation. Hydroxyurea, vincristine plus prednisolone, and imatinib brought about essentially no improvement in clinical condition and laboratory data. Although subsequent treatment with cytarabine (Ara-C) dramatically decreased the eosinophil count from 126 000/lL to 3700/lL, the patient’s left knee began to swell during the successful control of eosinophilia. Left knee biopsy revealed the recurrence of MFH just a week before her death due to respiratory failure. Because MFH is reported to cause neutrophilia through the production of interleukin (IL)-6 (1), we retrospectively measured the serum cytokine levels of this patient taken at the point of presentation of severe eosinophilia in March 2003. Both serum granulocytemacrophage colony-stimulating factor (GM-CSF) and IL-6 levels were high (118 and 31.0 pg/mL, respectively), although serum levels of other cytokines including IL-3, IL-5 and granulocyte colony-stimulating factor (G-CSF) were within the normal range. Immunohistochemical staining of the tumour specimen with anti-GM-CSF polyclonal antibody (N-19) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) confirmed the ectopic production of GM-CSF (Fig. 1C). These results indicated that intractable eosinophilia occurred because of the cytokine production by MFH in this patient. Although neutrophilia in MFH is not so rare, most cases produced IL-6 but not GM-CSF (2). Because production and survival of eosinophils are mainly regulated by IL-3, IL-5 and GM-CSF (3), production of GM-CSF in addition to IL-6 should induce marked eosinophilia as much as 200 000/lL in this case. This case indeed failed to fulfil the chronic eosinophilic leukaemia criteria of WHO classification because of the lack of increased count of myeloblasts (4). Therefore, presence of extremely high eosinophil count with immature morphology does not warrant autonomous proliferation. This report highlights the importance of considering occult or recurrent malignancies in patients with idiopathic hypereosinophilic syndrome (HES) even

Mononucleosis syndrome and acute monocytic leukemia

Abstract: The association of infectious mononucleosis and an immunocompromised host such as occurs in acute leukemia is reported. The most common cause of infectious mononucleosis is Epstein–Barr virus (EBV) and cytomegalovirus (CMV). Patients with mononucleosis syndrome caused by other agents are rare. We report a case of acute monocytic leukemia (AMoL) who developed varicella zoster virus (VZV) mononucleosis syndrome in the bone marrow recovery phase after myelosuppression due to high‐dose cytarabine. Mononuclear leukocytes appearing during the mononucleosis syndrome were very similar to the initial leukemic cells. Varicella zoster virus mononucleosis syndrome was confirmed by delayed herpes zoster rash with dermatomal distribution.