Makoto Sasaki

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.

Improvement of idiopathic thrombocytopenic purpura by antithyroid therapy

Abstract:  Here we report a case of idiopathic thrombocytopenic purpura accompanied by Graves’ disease. Improvement in thyroid function with methimazole led to the spontaneous recovery of the platelet count from 8 × 109/L to 84 × 109/L. Furthermore, the second fall and recovery of the platelet count well coincided with the recurrence of hyperthyroidism after the discontinuation of methimazole and its normalization by resumption of the drug, respectively. These parallel fluctuations of platelet and thyrotropin because of the cessation and resumption of antithyroid therapy suggests that correction of hyperthyroidism may be beneficial to the control of an imbalance in the immune system which impairs not only thyroid but also the platelet.

Spontaneous regression of natural killer cell lymphoma

Spontaneous tumour regression is extremely rare in aggressive lymphoma. A case of natural killer (NK) cell lymphoma with cutaneous manifestation showed an indolent clinical course, and the relapsed nodular lesion disappeared spontaneously without any treatment. Although only small number of T cells were present in the primary skin lesion, there was massive CD8-positive cytotoxic T cell infiltration in the relapsed lesion. This is believed to be the first report of an abscopal effect on NK cell lymphoma. Infiltration of cytotoxic T cells strongly suggests immunological attack against the lymphoma cells.

Frequent STAT3 activation is associated with Mcl-1 expression in nasal NK-cell lymphoma.

Nasal natural killer (NK)‐cell lymphoma was resistant to various antitumor agents. Although high expression of p‐glycoprotein has been reported, other molecular mechanism of the chemo‐resistance is largely unknown. Activation of STAT3 and expression of major apoptosis‐related proteins Bcl‐2, Bcl‐x, and Mcl‐1 were analyzed by immunohistochemistry. Effects of STAT3 inhibitor AG490 on NK‐YS cell line were analyzed by Western blotting and flow cytometric apoptosis assay. STAT3 was activated in six of the nine nasal NK‐cell lymphomas (67%). In contrast, STAT3 activation was detected in 35% of diffuse large B‐cell lymphoma (DLBCL) and in 10% of follicular lymphoma (FL). Frequent activation of STAT3 was significantly correlated with Mcl‐1 expression in nasal NK‐cell lymphoma, i.e., Mcl‐1 was positive in five of six STAT3‐active cases and negative in all three STAT3‐inactive ones. In DLBCL, not only six out of seven STAT3‐active cases (86%) but also eight out of thirteen STAT3‐inactive cases (62%) were positive for Mcl‐1 expression. Latent membrane protein‐1 was positive in four nasal NK‐cell lymphomas, among which three cases showed intermediate STAT3 activation. Inhibition of STAT3 activation by JAK inhibitor AG490 decreased Mcl‐1 expression and induced apoptosis in STAT3‐active NK‐YS cells. Serum starvation rather increased the Mcl‐1 level in NK‐YS cells, and this effect was also canceled by AG490. These results suggest that activation of STAT3‐Mcl‐1 axis may play a role in the chemotherapy resistance of nasal NK‐cell lymphoma. The pathway may be one of the future therapeutic targets of this intractable disease.

Expression levels of apoptosis-related proteins and Ki-67 in nasal NK / T-cell lymphoma

Objectives:  Nasal natural killer (NK)/T‐cell lymphoma is characterized by chemo‐resistance, angiodestruction, and aggressive tumor progression. Few studies exist on molecular characteristics of this disease entity.

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.

Low-dose doxorubicin-induced necrosis in Jurkat cells and its acceleration and conversion to apoptosis by antioxidants.

Summary. We treated rapidly growing Jurkat cells with 40 nmol/l of doxorubicin for 72 h. After 36 h, the G2‐arrested cells became larger and some of them started endoreplication. Nuclear staining with Hoechst 33342 combined with propidium iodide (PI) exclusion revealed that about 90% of the cells were necrotic at 72 h, although apoptotic cells accounted for only 8%. Incubation with 40 nmol/l of aclarubicin or cytosine β‐d‐arabinofuranoside for 60 h induced necrosis both in Jurkat and ml‐1 cells. Pre‐necrotic Jurkat cells incubated with 40 nmol/l of doxorubicin had much higher intracellular reactive oxygen species (ROS) levels than pre‐apoptotic ones. Addition of Tempol or Desferal accelerated doxorubicin‐induced necrosis and partially converted it into apoptosis. Both antioxidants reduced surviving colony numbers of prenecrotic Jurkat cells. n‐acetyl‐l‐cysteine had little effect on the apoptotic conversion but profoundly accelerated necrosis. Because an apoptosis‐resistant Jurkat subclone was also refractory to doxorubicin‐induced necrosis, apoptosis and necrosis might share some common pathways. Low‐dose doxorubicin increased micronuclei‐positive cell percentages and also suppressed high‐dose doxorubicin‐induced apoptosis in Jurkat and ml‐1 cells. Some of the prenecrotic cells, therefore, might survive and obtain genomic instability. Antioxidants may be useful to suppress, at least to some extent, this vicious consequence.

Activation of an ataxia telangiectasia mutation-dependent intra-S-phase checkpoint by anti-tumour drugs in HL-60 and human lymphoblastoid cells

In yeast cells, the intra‐S‐phase checkpoint slows down the rate of DNA replication in response to DNA damage. Here we showed that a similar checkpoint mechanism is present and activated by anti‐tumour drugs in HL‐60 and Epstein–Barr virus (EBV)‐transformed human lymphoblastoid cells. Using bromodeoxyuridine (BrdU) pulse labelling combined with two‐dimensional flow cytometric analysis, we clearly visualized the cell‐cycle progression of the BrdU‐positive population (cells originally belonging to the S phase) and detected even subtle changes in S‐phase progression induced by mild drug treatment conditions free of apoptosis. The DNA topoisomerase II inhibitors, doxorubicin and etoposide (250 nmol/l and 400 nmol/l, respectively, for 8 h), retained the BrdU‐positive HL‐60 cells in the latter half of S and G2/M positions, and the pyrimidine analogue anti‐metabolite, cytosine β‐ d‐arabinofuranose (Ara‐C; 50 nmol/l), kept them in early‐to‐late S phase after 8 h of incubation. Because 10 μmol/l of caffeine added 2 h later attenuated the S‐phase retardation by these drugs in HL‐60 cells, slowing of the S‐phase progression should be actively regulated. Furthermore, two ataxia telangiectasia (AT)‐derived lymphoblastoid cell lines were impaired in the doxorubicin‐induced S‐phase retardation, which indicated that the process is at least partially dependent on ataxia telangiectasia mutated (ATM) gene product. The inhibitory mechanism on S‐phase progression elicited by anti‐tumour drugs in HL‐60 and lymphoblastoid cells may therefore correspond to the intra‐S‐phase checkpoint of the yeast cells.

Prognostic significance of pleural or pericardial effusion and the implication of optimal treatment in primary mediastinal large B-cell lymphoma: a multicenter retrospective study in Japan.

The prognosis of patients with primary mediastinal large B-cell lymphoma has improved over recent years. However, the optimal treatment strategy including the role of radiotherapy remains unknown. We retrospectively analyzed the clinical outcomes of 345 patients with newly diagnosed primary mediastinal large B-cell lymphoma in Japan. With a median follow up of 48 months, the overall survival at four years for patients treated with R-CHOP (n=187), CHOP (n=44), DA-EPOCH-R (n=9), 2nd- or 3rd-generation regimens, and chemotherapy followed by autologous stem cell transplantation were 90%, 67%, 100%, 91% and 92%, respectively. Focusing on patients treated with R-CHOP, a higher International Prognostic Index score and the presence of pleural or pericardial effusion were identified as adverse prognostic factors for overall survival in patients treated with R-CHOP without consolidative radiotherapy (IPI: hazard ratio 4.23, 95% confidence interval 1.48–12.13, P=0.007; effusion: hazard ratio 4.93, 95% confidence interval 1.37–17.69, P=0.015). Combined with the International Prognostic Index score and the presence of pleural or pericardial effusion for the stratification of patients treated with R-CHOP without radiotherapy, patients with lower International Prognostic Index score and the absence of effusion comprised approximately one-half of these patients and could be identified as curable patients (95% overall survival at 4 years). The DA-EPOCH-R regimen might overcome the effect of these adverse prognostic factors. Our simple indicators of International Prognostic Index score and the presence of pleural or pericardial effusion could stratify patients with primary mediastinal large B-cell lymphoma and help guide selection of treatment.

More Than 13 Years of Hypereosinophila Associated With Clonal CD3−CD4+ Lymphocytosis Of TH2/TH0 Type

A 65-year-old Japanese woman was referred to our hospital because of hypereosinophilia lasting for more than 10 years, and skin ulceration, especially on the hands. Closer examination revealed the clonal proliferation of CD3-CD4+ T-lymphocytes. The patient had generalized pruritus without severe end-organ involvement and high serum levels of IgE. A diagnosis of monoclonal CD3-CD4+ T-lymphocyte-associated idiopathic hypereosinophilic syndrome (HES) was made based on these findings.This case showed that this newly recognized entity of HES is not restricted to Western countries. The abnormal T-cell clone was not merely Th2 type but was clearly Th2/Th0 type. Although this disease is considered prelymphoma, this patient did not develop lymphoma during more than 13 years of follow-up.Therefore, in some patients, clonal CD3-CD4+ lymphocyte-associated HES may take a more indolent course. In this subgroup, the control of clinical manifestations seems very important. In the present case, treatment with hydroxyurea quite dramatically improved the intractable skin manifestations, although the treatment lessened only the number of peripheral eosinophils and not the number of clonal CD3-CD4+ T-lymphocytes.