Kosei Arimura

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.

Successful treatment of Good syndrome with cytomegalovirus duodenoenteritis using a combination of ganciclovir and immunoglobulin with high anti-cytomegalovirus antibody titer

We describe the case of a 64-year-old woman with Good syndrome who presented with watery diarrhea and abdominal distention caused by cytomegalovirus (CMV) duodenoenteritis. Thymoma and hypogammaglobulinemia were first identified when the patient was 58 years old. She had repeatedly complained of symptoms even after thymectomy. Abdominal radiography revealed multiple air-fluid levels, and computed tomography revealed ascites and dilation of the small intestine. Immunofluorescent staining of specimens obtained by duodenal mucosal biopsy revealed intracellular inclusion bodies of CMV, although serum CMV pp65 antigenemia assays yielded negative results. CMV infection of the small intestine caused mucosal edema resulting in malabsorption. The patient was treated using ganciclovir and an immunoglobulin preparation with a high titer of antibodies against CMV (CMV-Ig), and subsequently made a rapid recovery from abdominal symptoms. When patients with Good syndrome complain of abdominal symptoms, particularly chronic diarrhea, a diagnosis of CMV gastroenteritis should not be excluded, even if negative results are obtained for CMV pp65 antigenemia assays. Combination therapy of ganciclovir and CMV-Ig seems useful for patients with CMV gastroenteritis.

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.

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.

True malignant histiocytosis with trisomy 9 following primary mediastinal germ cell tumor

A 24-year-old Japanese man was admitted due to bloody phlegm in May 2002. A diagnosis of mediastinal germ cell tumor, mixed type involving seminoma, immature teratoma and embryonal carcinoma, was made by transthoracic needle biopsy. Three months later, his complete blood counts revealed pancytopenia with high fever. Examination of bone marrow revealed increased atypical large histiocytes (5.6%) with hemophagocytosis, and thus, hemophagocytic syndrome related to germ cell tumor was diagnosed. In addition, chromosomal analysis of the bone marrow cells revealed a 47, XY, +9 genotype. Chemotherapies for germ cell tumor and hemophagocytic syndrome were performed without any improvement, and he died of diffuse alveolar damage. Autopsy revealed diffuse infiltration of immature histiocytes with hemophagocytosis in the liver, spleen and bone marrow. The atypical histiocytes were positive for CD68 and lysozyme and negative for lymphoid markers, and the diagnosis of true malignant histiocytosis associated with mediastinal germ cell tumor was made. The rare chromosomal abnormality of trisomy 9, a marker for benzene-related leukemia, was seen in the present case without apparent benzene exposure.

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.

Alteration of p16 (CDKN2) gene is associated with interleukin-2-induced tumor cell growth in adult T-cell leukemia.

Because tumorigenesis frequently involves the dysfunction of cell cycle-related proteins, we examined the effect of mutations in CDK inhibitor p16 and its linked genomic loci p15, cl.B, and 1063.7 on the growth of primary adult T-cell leukemia (ATL) cells. Southern blot analysis of primary ATL cells showed a significantly higher incidence of p16 gene alteration in acute ATL than in chronic ATL [67.7% (23/34) vs. 26.1% (6/23), respectively; p<0.003]. Similarly, polymerase chain reaction (PCR) analysis of p16 exon 2 revealed a higher incidence of alteration in acute ATL than in chronic ATL [52.9% (18/34) vs. 26.1% (6/23), respectively; p<0.05]. PCR-single strand conformation polymorphism analysis of exons 1 and 2 of p16 showed no mutations in the patients, with normal pattern by Southern blotting or PCR analysis. Notably five of six chronic ATL patients with abnormal p16 genes progressed to acute crisis within 4 months. PCR analysis of the p16 linked loci 1063.7, p15 exon 2, and cl.B found homozygous deletion in 55.9%, 20.6%, and 2.9% of acute ATL cells and 39.1%, 13.0%, and 0% of chronic ATL cells, respectively, showing no relationship of homozygous deletion in either loci with disease subtypes. In most cases, deletions were seen in multiple genes, including p16. Acute ATL cells had a higher frequency of multigene deletions than chronic ATL cells [44.1% vs. 17.4%; p<0.05]. When leukemic cells were analyzed for interleukin 2 (IL-2) responsive growth, only p16 gene alteration was directly associated with leukemic cell growth activity. Among leukemic cells showing high IL-2 responsiveness, 73.1% (19/26) had p16 gene alteration vs. 27.8% (5/18) of leukemic cells that showed low IL-2 responsiveness (p<0.005). p16 gene alteration was found in 73.3% (14/19) of leukemic cells showing high autonomous growth rates but in only 40.0% (10/25) of those leukemic cells showing low autonomous growth (p<0.03). These results suggest the following: alteration of p16-related genomic regions in ATL is usually a wide rearrangement including the p16 gene; within this region, only p16 gene alteration is associated with disease aggressiveness; and p16 gene deletion may be a proximate event in leukemogenesis.

Spontaneous regression associated with apoptosis in a patient with acute‐type adult T‐cell leukemia

We describe a 76‐year‐old man with acute‐type adult T‐cell leukemia, who demonstrated a spontaneous decrease in leukemic cell number, apparently coincident with apoptotic cell death. On admission the patients white blood cell count was 38.9 × 109/l with 77% abnormal lymphocytes. He also had hypoproteinemia (4.3 g/dl) from protein losing enteropathy. After admission the leukemic cell count decreased without chemotherapy, reaching 5.9 × 109/l after 2 months. Studies of peripheral lymphocytes demonstrated appearance of the apoptotic cells and DNA ladder formation from the beginning of regression. Same truncated proviral DNA was recognized in primary ATL cells through the whole clinical course. The hypoproteinemia improved with intravenous nutrition, followed by increase of the leukemic cells. This case is the first report that demonstrates tumor‐cell apoptosis induced clinical regression in adult T‐cell leukemia. Further, we speculate that the hypoproteinemia may have been involved in the leukemic cell apoptosis. Am. J. Hematol. 61:144–148, 1999.