Hideo Ohtsubo

NF-κB involvement in the activation of primary adult T-cell leukemia cells and its clinical implications

The HTLV-I provirus-encoded Tax protein induces NF-kappaB in Tax-transfected Jurkat T cells or HTLVL-I- infected T cells in vitro. Tax induction of NF-kappaB is presumed to be involved in proliferation and activation of primary leukemia cells in vivo. Recent studies have demonstrated that NF-kappaB activities in human T cells are mediated by at least four c-Rel-related DNA binding proteins - p50, p55, p75 and p85. We examined the significance of NF-kappaB induction in primary adult T cell leukemia cells and the induction kinetics of each of the four NF-kappaB species. Marked NF-kappaB activity was detected using an electrophoretic mobility shift assay (EMSA) in the primary cells of patients with acute disease, but little activity was noted in the cells of chronic patients. NF-kappaB activity was enhanced in a time-dependent manner in acute type cells cultured with mitogen-free medium; there was no induction of activity in chronic type cells. UV crosslinking demonstrated all four species of NFkappaB complex - high levels of p50 and lower levels of p55 and p75, in acute type cells; chronic type cells showed only the p50. As a control, normal resting T cells similarly showed only p50; control cells showed little change in activity when cultured without mitogenic stimulation, analogous to chronic type ATL. Northern blotting revealed enhancement of c-rel (encoding p85) and KBFI (encoding p50 and p55) expression in acute type cells during culture, while there was no significant enhancement of mRNAs in chronic type ATL cells or unstimulated normal T cells. Northern blotting also revealed that Tax is upregulated at the mRNA level in acute- but not chronic-type cells during culture. Expression of c-rel and KBF1 mRNAs in acute type cells appeared to be related to Tax mRNA expression. These results suggest that Tax is capable of inducing nuclear expression of all four NF-kappaB species in primary ATL cells of acute type patients, with marked effects on p55, p75, and p85. Tax induction of NF-kappaB species is regulated, at least in part, at a pretranslational level involving increases in c-rel and KBF1 mRNA.

Long-term Maintenance Combination Chemotherapy with OPECMPEC (Vincristine or Methotrexate, Prednisolone, Etoposide and Cyclophosphamide) or with Daily Oral Etoposide and Prednisolone Can Improve Survival and Quality of Life in Adult T-cell LeukemiaLymphoma

Acute leukemia and lymphoma varieties of adult T-cell leukemia/lymphoma (ATL) usually carry a poor prognosis. While etoposide is generally useful for treating ATL, especially as a daily oral maintenance regimen, etoposide has not proven effective in severe types of ATL efficient in some patients. Of 87 ATL patients whom we have treated, 51 had acute leukemia, 22 lymphoma and 14 progressive chronic leukemia. Seventy-nine patients were treated with a long term maintenance combination protocol, OPEC/MPEC (weekly doses of vincristine, 0.7 mg/m2 or methotrexate, 14 mg/m2; prednisolone, 20 mg/m2; etoposide, 70 mg/m2 and cyclophosphamide, 200 mg/m2). The other 8 patients, 3 with acute leukemia, 2 with lymphoma and 3 with progressive chronic leukemia, were treated with daily oral administration of 25 mg of etoposide and 10 mg of prednisolone (DOEP). The dose administered was modified in individual cases to maintain the granulocyte count and reduce the number of ATL cells. Considering both protocols, a complete response and a partial response were achieved in 31.0% and 58.6% patients, respectively. Median survival times (MST) of all patients and, acute leukemia, lymphoma and progressive chronic leukemia types were 7.5, 6.7, 9.6 and 12.4 months, respectively. Respective MST of patients treated with OPEC/MPEC or DOEP protocols were 7.1 and 18.0 months. Relatively normal WBC counts, lower lactate dehydrogenase concentration and normal calcium concentration, limited numbers of anatomic sites involved, good performance status and good response to chemotherapy were significantly associated with long survival time. Drug toxicity was not apparent, and about half of patients were treated in an outpatient setting.

Granulocyte‐colony stimulating factor‐induced proliferation of primary adult T‐cell leukaemia cells

Granulocyte‐colony stimulating factor (G‐CSF) is known to induce proliferation and differentiation of granulocyte progenitors, and is widely used to treat neutropenia induced by intensive chemotherapy for malignant lymphoma or adult T‐cell leukaemia/lymphoma (ATL). G‐CSF is thought not to stimulate malignant lymphoid cells. In the present study we examined the ability of G‐CSF to induce in vitro growth of primary ATL cells from 14 patients (nine acute‐type, two chronic‐type and three lymphoma‐type), and we analysed the in vivo counts of ATL cells in patients who received G‐CSF for neutropenia. FACS analysis using phycoerythrin‐labelled recombinant G‐CSF demonstrated that ATL cells from 11/14 patients express some G‐CSF receptor (G‐CSFR), with a range between 5.4% and 87.3%. Cells expressing G‐CSFR also expressed CD4. Reverse polymerase chain reaction (PCR) analysis demonstrated expression of G‐CSFR messenger RNA in G‐CSFR expressing cells. Leukaemic cells derived from seven (four acute‐type, one chronic‐type and two lymphoma‐type) of the 14 patients proliferated in vitro in response to G‐CSF, as measured by [3H]thymidine incorporation; maximum responses were at G‐CSF concentrations of 10–100 ng/ml. Nine of 14 patients receiving rG‐CSF for neutropenia were analysed retrospectively for ATL cell numbers. Four patients whose primary tumour cells proliferated in response to rG‐CSF in vitro showed a significant increase in ATL cell count after administration of rG‐CSF (P =0.038), whereas five patients whose leukaemic cells did not proliferate in vitro showed no significant increase in ATL cell count. G‐CSF can stimulate proliferation of ATL cells which may complicate therapy for this disease.

Matrix metalloproteinase inhibitor reduces apoptosis induction of bone marrow cells in MDS‐RA

Abstract:  Background and objectives: We examined the involvement of apoptosis with myelodysplastic syndrome (MDS) accompanied by peripheral cytopenias despite normo‐hypercellular bone marrow. Materials and methods: Bone marrow smears from 31 patients with MDS‐refractory anemia (RA) and five normal controls were stained using the in situ end labeling (ISEL) method. Next, the inhibitory effects of a caspase‐3 inhibitor, matrix metalloproteinase inhibitor (MMPI), anti‐tumor necrosis factor (TNF)‐α or anti‐Fas antibody upon the apoptosis induction in overnight cultures of bone marrow cells from the patients were examined. Further, TNF‐α, transforming growth factor (TGF)‐β and soluble Fas ligand (sFasL) concentrations in culture supernatants of the cells were assessed by enzyme‐linked immunosorbent assay (ELISA). Results: The incidence of ISEL‐positive cells among MDS patients was significantly higher than in normal controls (50.8 ± 14.0% vs. 11.3 ± 2.4%; P < 0.0001). A caspase‐3 inhibitor reduced significantly the ISEL‐positive rates (32.6 ± 15.2% vs. 50.2 ± 16.5%; P < 0.0001). Anti‐TNF‐α or anti‐Fas antibody reduced the ISEL‐positive rates significantly (28.2 ± 6.0%, 29.2 ± 5.8%, vs. 44.2 ± 3.4%, P < 0.001, P = 0.001, respectively). KB‐R7785 also significantly decreased the ISEL‐positive rates (18.0 ± 9.3% vs. 43.6 ± 14.0%; P < 0.0001). The concentration of TNF‐α was significantly reduced by KB‐R7785 (P < 0.05), whereas that of TGF‐β was not. Concentration of sFasL was under detectable level in the present assay system. The derivatives of KB‐R7785 that can be administrated orally showed inhibitory effect on apoptosis induction as well. Conclusions: These findings suggest that MMPIs inhibits the apoptosis induction of MDS bone marrow cells via the inhibition of TNF‐α and probably sFasL secretion, and that MMPIs can be used to control the abnormal induction of apoptosis in MDS.

Severe autoimmune thrombocytopenic purpura during interferon-alpha therapy for chronic myelogenous leukemia.

Interferon (IFN)-α is a leukocyte-derived cytokine and is used to treat several hematopoietic malignancies. The most common adverse effects of IFN-α are flu-like symptoms and usually insignificant. However, adverse effects due to autoimmune mechanisms are often hazardous and irreversible, although their frequency is low. In the present report, we describe a 55-year-old female with chronic myelogenous leukemia who developed severe autoimmune thrombocytopenia during IFN-α therapy. The lowest platelet count was 6 × 109/l with severe hemorrhagic tendency. The present report strongly suggests the clinical importance of autoimmune thrombocytopenia as an adverse effect of IFN-α.

Epstein-Barr virus involvement in T-cell malignancy: significance in adult T-cell leukemia.

Epstein-Barr virus (EBV) was first reported as the causative virus of Burkitts lymphoma in 1964. Since then, EBV has also been associated with infectious mononucleosis, AIDS and transplant-related B cell lymphomas, and nasopharyngeal cancer. The virus has further been linked with T cell lymphomas, Hodgkin disease, and NK leukemia or LGL leukemia, establishing a concept of a wide spectrum of EBV associated malignant disorders. EBV DNA encodes several proteins such as EBNA1-6, LMP 1, 2 and others. Recent studies have demonstrated that EBNA2, EBNA5, EBNA3A, EBNA 3C are essential for transformation, and that any gene product is not sufficient to transform cells by itself. Further there are different mechanisms of virus-associated transformation or carcinogenesis among EBV-associated malignant disorders. On the other hand, human T lymphotropic virus type I (HTLV-I) is known as a causative virus of adult T cell leukemia (ATL). However, precise molecular mechanisms of leukemogenesis in ATL still remains unclear. Some additional factors to HTLV-I infection are supposed to be involved in complete leukemogenesis. We demonstrated that HTLV-I infected T cells and primary ATL cells express EBV receptor/CD21 on the cell surface. Therefore, it is possible that EBV infection is one of the factors. We further investigated this possibility in 6 HTLV-I infected T cell lines and primary ATL cells from 18 patients with ATL. However, no EBV genome was detected in both T cell lines and primary ATL cells. EBV involved T-cell lymphoma has unique clinical manifestations as compared to non-EBV involved T-cell lymphoma. Therefore, it is still possible that a small group of ATL patients with unique clinical manifestations is associated with EBV.

IL-2-induced growth of CD8+ T cell prolymphocytic leukemia cells mediated by NF-κB induction and IL-2 receptor α expression

Abstract The binding of interleukin-2 (IL-2) to its receptor on normal T cells induces nuclear expression of nuclear factor κB (NF-κB), activation of the IL-2 receptor (IL-2R) α chain gene, and cell proliferation. In the present study, the role of IL-2R signaling in the growth of CD8 + T cell prolymphocytic leukemia (T-PLL) cells has been investigated. Flow cytometry revealed that primary leukemia cells from a patient with CD8 + T-PLL expressed IL-2Rα and β chains, and the cells showed a proliferative response and an increase in IL-2Rα expression on culture with exogeneous IL-2. Northern blot analysis failed to detect IL-2 mRNA, suggesting that IL-2 may act in a paracrine manner in vivo . Electrophoretic mobility-shift assays revealed that recombinant IL-2 increased NF-κB binding activity in nuclear extracts of the leukemia cells, and Northern blot analysis showed that IL-2 increased the abundance of mRNAs encoding the NF-κB components c-Rel and KBF1 in these cells. IL-2 binding analysis demonstrated that IL-2 markedly increased the number of low affinity IL-2Rs on the leukemia cells, without an effect on the number of high-affinity IL-2Rs. These results show that IL-2 is capable of inducing the nuclear expression of NF-κB in primary CD8 + T-PLL cells, and that this effect is mediated, at least in part, at a pretranslational level.

Human T-cell lymphotropic virus type I Tax activates lung resistance-related protein expression in leukemic clones established from an adult T-cell leukemia patient

OBJECTIVE We examined the significance of human T-cell lymphotropic virus type I (HTLV-I) Tax protein-induced resistance to anticancer drugs and the relationship between Tax and multidrug resistance proteins. MATERIALS AND METHODS S1T cell, a leukemic non-Tax-producing T-cell clone established from an adult T-cell leukemia (ATL) patient, S1TcTax05 and S1TcTax10 clones, transfected with Tax stably expressing cDNA, and S1Tneo, transfected with a neomycin-resistant gene, were examined for Tax-related anticancer drug resistance. Resistance of those cells to the anticancer drugs doxorubicin, etoposide, cisplatin, and vindesine was tested with the MTT method. Expression of multidrug resistance protein mRNAs (MDR1, MRP1, cMOAT/MRP2, and LRP) was analyzed with reverse transcriptase polymerase chain reaction (RT-PCR). Doxorubicin subcellular distribution in those cells was examined by fluorescence microscopy. RESULTS S1TcTax05 and S1TcTax10 showed resistance to doxorubicin, etoposide, and vindesine, but not to cisplatin as compared with S1T or S1Tneo. RT-PCR demonstrated that MRP1 mRNA was expressed, but MDR1, cMOAT, and LRP mRNAs were not in S1T or S1Tneo. Marked expression of LRP mRNA was detected, but no change of MDR1, MRP1, or cMOAT mRNA expression in Tax-expressing S1TcTax05 and S1TcTax10. Fluorescence microscopy demonstrated that doxorubicin was distributed mainly in the cytoplasm of S1TcTax05 and S1TcTax10, and in the nucleus of S1T and S1Tneo. CONCLUSIONS These findings suggest that Tax-related drug resistance of ATL cells is due to LRP and not MDR1, as reported previously. These findings in cells derived from an ATL patient suggest a novel mechanism for drug resistance in Tax-expressing ATL cells.

Cytomegalovirus infection is not necessarily a poor prognostic factor in adult T-cell leukemia/lymphoma.

The relationship between cytomegalovirus (CMV) antigenemia and the clinical course was examined in 57 patients with adult T‐cell leukemia/lymphoma (ATLL). All patients included had the acute/lymphoma type of ATL according to the criteria of the Japan Lymphoma Study Group (LSG). CMV antigenemia was assessed on admission and at the time when the patients had fever higher than 37.5°C, which did not respond to antibiotics for longer than 3 days. The incidence of CMV antigenemia was 44%. Approximately 90% of patients with CMV antigenemia died of infections with viruses, bacteria, and/or fungi, while approximately 40% of patients without CMV antigenemia died of deterioration of ATLL (P<0.0001). In this study, the patients with CMV antigenemia tended to survive longer than those negative for it (321.4 days vs. 266.2 days), although there was no statistical significance (P=0.09). Kaplan‐Meier analysis revealed that CMV antigenemia was not a poor prognostic factor. When the disease status of ATLL was evaluated by thymidine kinase (TK) and soluble interleukin 2 receptor (sIL‐2R), both had lower titers during CMV antigenemia (TK: P=0.01, sIL‐2R: P=0.03, respectively). Therefore, CMV infections in ATLL patients seemed to have bimodal meanings; CMV infection at the end of clinical course were life‐threatening, but infection during the first half of clinical course seemed to suppress ATLL activity and to contribute to the longer survival of the patients. J. Med. Virol. 62:140–143, 2000.

Interferon-α Therapy following Autologous Peripheral Blood Stem Cell Transplantation for Adult T Cell Leukemia/Lymphoma

In the present report, we describe a case of adult T cell leukemia/lymphoma (ATLL), a 58-year-old woman, successfully treated with interferon (IFN)-α following autologous peripheral blood stem cell transplantation (auto-PBSCT). The patient remains in remission with full performance status for more than 12 months. Auto-PBSCT reduced the abdominal lymphoma mass and IFN-α eliminated residual tumor cells, possibly through the induction of specific T-cell subsets expressing CD3, CD8 on their surfaces and either IFN-γ or tumor necrosis factor (TNF)-α in cytoplasm. We have treated a total of 4 ATLL patients with auto-PBSCT, including the case presented herein. All other patients treated with auto-PBSCT were not followed by adjuvant chemotherapy or cytokine therapy and relapsed within 3 months after auto-PBSCT. This evidence suggests that the therapeutic success of the present case was attributable to the administration of IFN-α immunotherapy following auto-PBSCT.