Toshiyuki Takagi

NELF, a multisubunit complex containing RD, cooperates with DSIF to repress RNA polymerase II elongation.

DRB is a classic inhibitor of transcription elongation by RNA polymerase II (pol II). Since DRB generally affects class II genes, factors involved in this process must play fundamental roles in pol II elongation. Recently, two elongation factors essential for DRB action were identified, namely DSIF and P-TEFb. Here we describe the identification and purification from HeLa nuclear extract of a third protein factor required for DRB-sensitive transcription. This factor, termed negative elongation factor (NELF), cooperates with DSIF and strongly represses pol II elongation. This repression is reversed by P-TEFb-dependent phosphorylation of the pol II C-terminal domain. NELF is composed of five polypeptides, the smallest of which is identical to RD, a putative RNA-binding protein of unknown function. This study reveals a molecular mechanism for DRB action and a regulatory network of positive and negative elongation factors.

Evidence that P-TEFb alleviates the negative effect of DSIF on RNA polymerase II-dependent transcription in vitro.

Recently, a positive and a negative elongation factor, implicated in 5,6‐dichloro‐1‐β‐D‐ribofuranosylbenzimidazole (DRB) inhibition of transcription elongation, has been identified. P‐TEFb is a positive transcription elongation factor and the DRB‐sensitive kinase that phosphorylates the C‐terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II). PITALRE, a member of the Cdc2 family of protein kinases, is the catalytic subunit of P‐TEFb. DSIF is a human homolog of the yeast Spt4‐Spt5 complex and renders elongation of transcription sensitive to DRB. DRB sensitivity‐inducing factor (DSIF) binds to RNA Pol II and may directly regulate elongation. Here we show a functional interaction between P‐TEFb and DSIF. The reduction of P‐TEFb activity induced by either DRB, antibody against PITALRE, or immunodepletion resulted in a negative effect of DSIF on transcription. DSIF acts at an early phase of elongation, and the prior action of P‐TEFb makes transcription resistant to DSIF. The state of phosphorylation of CTD determines the DSIF‐RNA Pol II interaction, and may provide a direct link between P‐TEFb and DSIF. Taken together, this study reveals a molecular basis for DRB action and suggests that P‐TEFb stimulates elongation by alleviating the negative action of DSIF.

Structure and Function of the Human Transcription Elongation Factor DSIF

5,6-Dichloro-1-β-d-ribofuranosylbenzimidazole (DRB) is a classic inhibitor of transcription elongation by RNA polymerase II (pol II). We have previously identified and purified a novel transcription elongation factor, termed DSIF (for DRBsensitivity-inducing factor), that makes transcription sensitive to DRB. DSIF is composed of 160- and 14-kDa subunits, which are homologs of the Saccharomyces cerevisiae transcription factors Spt5 and Spt4. DSIF may either repress or stimulate transcription in vitro, depending on conditions, but its physiological function remains elusive. Here we characterize the structure and function of DSIF p160. p160 is shown to be a ubiquitous nuclear protein that forms a stable complex with p14 and interacts directly with the pol II largest subunit. Mutation analysis of p160 is used to identify structural features essential for its in vitro activity and to map the domains required for its interaction with p14 and pol II. Finally, a p160 mutant that represses DSIF activity in a dominant-negative manner is identified and used to demonstrate that DSIF represses transcription from various promoters in vivo.

Lenalidomide plus dexamethasone treatment in Japanese patients with relapsed/refractory multiple myeloma

We conducted a multicenter, open-label study to investigate the safety, efficacy, and pharmacokinetics of lenalidomide in Japanese patients with relapsed or refractory multiple myeloma The study was composed of the “monotherapy phase”, a dose-escalation phase, to determine the tolerability to single agent lenalidomide and the “combination phase” to determine the safety and obtain preliminary data on the efficacy of lenalidomide plus dexamethasone. The primary end points were the tolerability to 25xa0mg lenalidomide and safety. Nine and six patients were enrolled in the monotherapy phase and the combination phase, respectively. Since 25xa0mg of monotherapy treatment did not satisfy the DLT criteria, this dose was employed in the combination phase. The major adverse event was myelosuppression. At the planned interim analysis (median study duration, 26.3xa0weeks), grade 3 or grade 4 neutropenia was observed with high frequency (66.7%). However, all adverse events observed were clinically manageable. In the combination cohort, the overall response rate (≥PR) was 100%. The pharmacokinetics of lenalidomide showed rapid absorption and elimination after both single and multiple doses. In conclusion, 25xa0mg of lenalidomide was given safely as a single agent or in combination with dexamethasone in Japanese patients. The good efficacy of the combination therapy was also demonstrated in this study.

Japanese multicenter phase II study of CHOP followed by radiotherapy in stage I–II1, diffuse large B-cell lymphoma of the stomach

CHOP (cyclophosphamide, doxorubicin, vincristine and prednisone) followed by radiotherapy is regarded as standard care for localized aggressive lymphoma; however, prospective confirmation of its applicability to localized primary gastric lymphoma is inadequate, and most patients in Japan have been initially treated with gastrectomy. We conducted a multicenter phase II study to evaluate the feasibility and efficacy of the non‐surgical treatment. Eligibility criteria required primary gastric diffuse large B‐cell lymphoma, stage I–II1, age 20–75, performance status 0–1 and adequate organ function. Treatment consisted of three cycles of CHOP followed by radiotherapy 40.5 Gy. Fifty‐five patients were enrolled between December 1999 and February 2003, and 52 eligible patients were analyzed. Patient characteristics were as follows: median age, 61 years; 28 men, 24 women; 36 with stage I, 16 with stage II1; 47 with a low International Prognostic Index (IPI) and five with a low–intermediate IPI. All but one patient completed planned treatment. No serious complications including massive hemorrhage or perforation were observed. A complete response was achieved in 48 of the 52 patients (92%, 95% confidence interval: 82–98%) and progressive disease in three. Two patients underwent salvage gastrectomy due to disease persistence or recurrence. With a median follow‐up period of 28 months, 2‐year progression‐free and overall survivals were 88 and 94%, respectively. CHOP followed by radiotherapy is safe and highly effective in localized gastric diffuse large B‐cell lymphoma. This organ‐preserving treatment should be considered as a very reasonable therapeutic option. (Cancer Sci 2005; 96: 349–352)

Low-dose thalidomide plus low-dose dexamethasone therapy in patients with refractory multiple myeloma

We report the results of a non‐randomized phase II study of low‐dose thalidomide plus low‐dose dexamethasone therapy in 66 patients with refractory multiple myeloma. The overall response rate (near complete, partial and minimal response) was 63.6%, and progression‐free and overall survival periods were 6.2 and 25.4u2003months. In adverse events, the incidence of peripheral neuropathy and deep vein thrombosis was lower than the data reported in USA and Europe. On the other hand, leukopenia was observed in 41% of patients, including 11% of those with Grade 3. Leukopenia was closely related to pretreatment pancytopenia, especially thrombocytopenia. The incidence of adverse events related to dexamethasone was low. In conclusion, low‐dose thalidomide plus low‐dose dexamethasone therapy was as effective as high‐dose thalidomide plus high‐dose dexamethasone therapy in patients with refractory multiple myeloma. Leukopenia is one of the most serious adverse events in Japanese patients, especially in patients with pretreatment pancytopenia.

A survey of the potency of Japanese illicit cannabis in fiscal year 2010

In recent years, increased cannabis potency, or Δ(9)-tetrahydrocannabinol (THC) content in cannabis products, has been reported in many countries. A survey of Japanese illicit cannabis was conducted from April 2010 to March 2011. In Japan, all cannabis evidence is transferred to the Minister of Health, Labour and Welfare after criminal trials. The evidence was observed at Narcotics Control Department offices in major 11 cities. The total number of cannabis samples observed was 9072, of which 6376 were marijuana. The marijuana seizures were further classified, and it was found that in terms of the number of samples, 65.2% of them were seedless buds, and by weight 73.0% of them were seedless buds. Seedless buds were supposed to be sinsemilla, a potent class of marijuana. THC, cannabinol (CBN) and cannabidiol (CBD) in marijuana seizures exceeding 1g were quantified. The number of samples analyzed was 1115. Many of them were shown to contain CBN, an oxidative product from THC. This was a sign of long-term storage of the cannabis and of the degradation of THC. Relatively fresh cannabis, defined by a CBN/THC ratio of less than or equal to 0.1, was chosen for analysis. Fresh seedless buds (335 samples) contained an average of 11.2% and a maximum of 22.6% THC. These values are comparable to those of high potency cannabis as defined in previous studies. Thus, this study shows that highly potent cannabis products are distributed in Japan as in other countries.

Successful treatment by azacitidine therapy of intestinal Behçet’s disease associated with myelodysplastic syndrome

Myelodysplastic syndrome (MDS) is a heterogeneous group of clonal hematopoietic stem cell diseases. It has been reported that several autoimmune diseases are associated with MDS. Recently, the co-occurrence of MDS with trisomy 8 and rare disorders of the immune system, such as Behçet’s disease (BD), has been described. Prognosis in the older-onset group of MDS-associated BD is unfavorable. Here, we report a case of MDS-associated intestinal BD treated successfully by azacitidine therapy. A 59-year-old Japanese male suffering from recurrent high fever, melena, and oral and genital ulcerations was diagnosed with MDS with trisomy 8 and intestinal BD by endoscopic and bone marrow examinations. Immunosuppressive therapies, including infliximab, were ineffective. Due to his severe emphysema, the patient was considered ineligible for stem cell transplantation, and azacitidine therapy was initiated. With the exception of fever, the symptoms of intestinal BD improved, and severe malnutrition and anemia were ameliorated. Fluorescence in situ hybridization analyses of the bone marrow before the eighth cycle revealed that the trisomy 8 had not decreased. To our knowledge, this is the first report of azacitidine therapy for MDS-associated BD. We suggest that azacitidine may control intestinal BD by mechanisms other than those responsible for its effect in MDS.

Primary Non-Hodgkin's Lymphoma of the Male Breast: A Case Report.

A 69-year-old Japanese man presented with bilateral gynecomastia and a soft, mobile and clearly defined mass beneath the left nipple. A round radiopaque mass was revealed on mammography. The tumor was homogeneous, hypoechoic and measured 2.4X3.9 cm on ultrasonography. Based on a diagnosis of malignant lymphoma by needle aspiration cytology, a modified radical mastectomy with ipsilateral axillary lymph node dissection was performed. Malignant diffuse large B-cell type lymphoma was diagnosed histologically. Whole body examinations revealed no evidence of other tumors. Three courses of adjuvant CHOP therapy were subsequently performed. The patient is free of recurrence 12 months after surgery.Primary non-Hodgkin’s lymphoma (NHL) of the male breast is extremely rare. The occurrence of lymphoma in this patient could be related to elevated estrogen levels.

Successful treatment of Philadelphia chromosome-positive mixed phenotype acute leukemia by appropriate alternation of second-generation tyrosine kinase inhibitors according to BCR - ABL1 mutation status

Philadelphia chromosome-positive mixed phenotype acute leukemia (Ph+MPAL) is a rare type of acute leukemia having myeloid and lymphoid features. In the present study, we describe the successful treatment of a 71-year-old Japanese female patient with Ph+MPAL by the alternation of second-generation tyrosine kinase inhibitors according to BCR-ABL1 mutations. The patient survived in her third complete remission (CR) for over 4xa0years. In her first CR, the patient was treated with multiple-agent chemotherapy and underwent maintenance therapy with imatinib and monthly vincristine and prednisolone (VP). At the first relapse, an examination of the bone marrow revealed a transformation into acute lymphoblastic leukemia and an F317L mutation in BCR-ABL1 gene, which responded preferentially to nilotinib over dasatinib. She achieved second CR, and nilotinib with VP therapy was selected for maintenance treatment. At second relapse, BCR-ABL1 mutational analysis revealed Y253H mutation instead of F317L mutation, resulting in resistance to nilotinib. The patient achieved third CR with dasatinib and VP therapy, and maintained CR with this treatment. This suggests that appropriate alternation of TKIs may contribute to long-term survival in elderly patients with Ph+MPAL.