tBID, a membrane-targeted death ligand, oligomerizes BAK to release cytochrome c.

TNFR1/Fas engagement results in the cleavage of cytosolic BID to truncated tBID, which translocates to mitochondria. Immunodepletion and gene disruption indicate BID is required for cytochrome c release. Surprisingly, the three-dimensional structure of this BH3 domain-only molecule revealed two hydrophobic alpha-helices suggesting tBID itself might be a pore-forming protein. Instead, we demonstrate that tBID functions as a membrane-targeted death ligand in which an intact BH3 domain is required for cytochrome c release, but not for targeting. Bak-deficient mitochondria and blocking antibodies reveal tBID binds to its mitochondrial partner BAK to release cytochrome c, a process independent of permeability transition. Activated tBID results in an allosteric activation of BAK, inducing its intramembranous oligomerization into a proposed pore for cytochrome c efflux, integrating the pathway from death receptors to cell demise.

A neuron-specific splicing switch mediated by an array of pre-mRNA repressor sites: evidence of a regulatory role for the polypyrimidine tract binding protein and a brain-specific PTB counterpart.

Tissue- and stage-specific alternative splicing events are widespread in mammals, yet the factors and mechanisms that direct these important posttranscriptional events are poorly understood. In this study, we focus on the 24-nt exon of the GABA(A) receptor gamma2 pre-mRNA, which is subject to neuron-specific and developmental splicing regulation in the rat cerebellum. Here we show biochemical evidence for a mechanism that directs the selective repression of the neuron-specific exon in non-neuronal splicing extracts derived from HeLa cells. Key evidence includes the discovery that the pathway of gamma2 pre-mRNA splicing switches from exon skipping to exon selection in splicing reactions with a short RNA competitor containing the 3' splice site region upstream of the 24-nt exon. In this assay, exon selection results from the coordinate activation of both flanking introns. A detailed dissection of this pre-mRNA region shows that it contains four repressor sites clustered around the branch site and extending into the 24-nt exon. These repressor sites are pyrimidine rich and bind avidly to the polypyrimidine tract binding protein (PTB) in HeLa nuclear extracts as determined by UV crosslinking/competition assays. Repression of the exon selection pathway is closely associated with the appearance of a specific RNA-protein complex, indicative of an inhibitor complex, that assembles on the repressor array. Upon the switch to the exon selection pathway, a substantial decrease in the inhibitor complex and a reciprocal increase in spliceosome complex A is observed. Excess recombinant PTB squelches the splicing switch and reestablishes exon skipping as the predominant splicing pathway. Extracts prepared from rat brain nuclei show reduced levels of conventional PTB compared to other splicing factors. Nonetheless, the rat brain nuclear extracts contain an activity that assembles an analogous inhibitor complex efficiently. We report a 59-kDa protein, p59, which has an electrophoretic mobility distinct from HeLa and rat kidney PTB, and which behaves in RNA binding assays as if it is the PTB counterpart in rat brain. Evidence that rat brain p59 is structurally related to PTB stems from western blot and immunoprecipitation analysis with a monoclonal antibody specific for the hnRNP I isoform of PTB. A model describing how the repressor array directs coordinate splicing regulation of flanking introns in the context of overlapping positive regulatory elements is discussed. The sequence, (5') UUCUCU (3'), in a pyrimidine context is associated with one class of intron splicing repressor sites that binds PTB in a variety of pre-mRNAs that are regulated by tissue-specific programs.

Essential nucleotides direct neuron-specific splicing of gamma 2 pre-mRNA.

Tissue-and stage-specific pre-mRNA splicing events are prevalent in mammals, yet molecular details are lacking about these important mechanisms of posttranscriptional gene control. In this study, we investigate the regulated splicing of rat gamma 2 pre-mRNA, a subunit of the GABAA receptor, as a step toward understanding the molecular basis of a neuron-specific splicing event involving cassette exon selection. Cell-and substrate-specific regulation of gamma 2 pre-mRNA is recapitulated in a neuronal cell line derived from the cerebellum, which produces enhanced levels of the exon-selected mRNA. In contrast, a control cell line derived from non-neuronal cells of the pituitary produces prominent levels of the unregulated, exon-skipped mRNA. The cerebellar and pituitary cell lines are well matched in overall splicing efficiency and produce an invariant pattern of splicing for a control substrate, which is alternatively spliced but not regulated in this system. The appropriateness of the two cell lines is indicated by an extended mRNA mapping experiment, which documents the region-specific switch in exon selection throughout rat brain. Using this pair of cell lines, we show that large intron segments flanking the regulated exon are dispensable for regulation. These intron regions have been deleted to generate a minimal splicing substrate for the purpose of identifying essential RNA elements. In this context, we show that essential nucleotides are located at positions +7, +8, and +9 of the regulated exon and in a 9-nt adenosine-rich region of the adjacent 3' splice site. Due to the proximity and base complementarity of the required nucleotides, experiments were devised to test models involving the recognition of two single-stranded signals, or one duplex RNA signal. These results clearly disfavor the duplex RNA recognition model and indicate that the required regions are recognized as independent, single strands in neuronal cells. A weak 5' splice site adjacent to the regulated exon is required as a third essential element. Although the importance of a weak 5' splice site is common to other regulated systems such as NCAM, the essential nucleotides in the exon and 3' splice site region defined in this study for gamma 2 splicing regulation are novel.

Regulated splicing of gamma2 pre-messenger RNA in neuronal cells.

The pre-mRNA encoding the gamma 2 subunit of the gamma-aminobutyric acid Type A receptor is spliced in a tissue-specific manner in mammalian brain resulting in mRNAs containing or lacking a 24 nucleotide exon, gamma 2L and gamma 2S, respectively. The gamma 2S mRNA predominates in pituitary, whereas the gamma 2L predominates in brainstem, spinal cord and cerebellum. In this study, a cell line derived from rat cerebellum that qualitatively reproduces regulated splicing of gamma 2 pre-mRNA was identified and used to dissect cis-regulatory elements. Sequence elements that alter the selection of the 24 nucleotide exon fall into two functional classes-activating elements and inhibitory elements. We identified several inhibitory elements that inhibit splicing of the 24 nucleotide exon in all cell types as well as specific inhibitory elements which repress splicing in non-neuronal cells. Activating elements are localized within conserved intron regions, as judged by a comparison of rat and human gene sequences, and appear to function generally in activating splicing of the 24 nucleotide exon in the cell types tested so far. These results are compatible with a hypothesis in which the mechanism of regulation involves a release from inhibition. Current experiments are aimed toward the development of tools to identify the trans-regulatory components.

[Cerebral and testicular myeloblastoma formation in relapsing acute myeloid leukemia (M1) with t(8;21)].

This paper reports a relapsed case of acute myeloid leukemia with intracranial, testicular and intestinal tumor formation. A-56-year-old male, diagnosed as M1 on September, 1988, entered complete remission on October 14, 1988, aided by JAL-SG and AML-85 regimen. Blast cells with Auer rods demonstrated 8;21 translocation lacking 11q with 30 of 30 analyzed bone marrow cells, and the following antigen pattern: CD5+, CD19+, CD33+, CD56+, HLA-DR+. After 4 courses of post remission therapy, the maintenance therapy was discontinued because of his liver dysfunction. He was discharged on May, 1989, and was seen as an out patient. He complained of left hemiplegia and was re-admitted on September 30, 1989. Though the bone marrow was in complete remission on September 4th, CT scan and MRI demonstrated intracranial tumor formation. Bone marrow relapse occurred on October 27th, eventually resulting in his death on November 18th. Autopsy showed intracranial, testicular and intestinal tumor formation and blast cell invasion into the liver, spleen and kidneys. We analyzed the characteristics of 14 cases with intracranial tumor formation previously reported. The focal neurological symptoms reflecting the intracranial tumor mass effect were considered to be important initial signs. CT scan was a useful tool for diagnosis. The average age of the 14 cases was 38, 9 and the male/female ratio was 9:5. Six of 9 cases, diagnosed by FAB classification, were M2 and one of the 6 cases in whom chromosomes of blast cells were examined had t(8;21). Though irradiation seemed effective for the reduction of tumor mass, the patients' prognosis was poor.(ABSTRACT TRUNCATED AT 250 WORDS)

[Complete remission with MEC regimen of acute myeloid leukemia (M4) secondary to 5-year treatment of non-Hodgkin lymphoma].

A 32-year-old woman was admitted to our hospital with pyrexia and general lymphadenopathy in July 1984. She was diagnosed as having malignant lymphoma (follicular, small cleaved cell), stage IV based on the histological findings of lymph nodes in the neck and bone marrow specimen. She was treated with melphalan orally for 3 years, followed by MACOP-B. She attained partial remission with MACOP-B. Thereafter, she received melphalan or Endoxan orally as maintenance therapy. She developed fever and swelling in the gingivae in October 1989. Peripheral blood showed WBC 80,200/microliters with 7.5% myeloblasts and 85.5% monocytes. Bone marrow aspirate revealed hypercellularity with 47.9% myeloblasts, 46.5% monoblasts and monocytes, which were positive for peroxidase and NSE stains. The karyotype of bone marrow cells showed a 46,XX,t(9;11). The lysozyme in serum was elevated. She was diagnosed having AML (M4). DCMP regimen was initiated but failed to achieve CR. Consequently she received MEC regimen and obtained complete remission, lasting for 6 months. Patients with second leukemia have a low probability of achieving complete remission using conventional chemotherapy. The MEC regimen is thought to be one of the most promising treatments for secondary leukemia.

Treatment of diffuse non-Hodgkin's lymphoma with combined chemotherapy using methotrexate, adriamycin, cyclophosphamide, vincristine, prednisolone and bleomycin (MACOP-B).

Forty-nine previously untreated adult patients with diffuse non-Hodgkin's lymphoma were treated with MACOP-B (methotrexate, adriamycin, cyclophosphamide, vincristine, prednisolone and bleomycin) between December 1986 and December 1990. Forty patients (82%) achieved a complete response (CR), three (6%) a partial response (PR), while four (8%) had either no response or progression of disease, one (2%) patient ceased MACOP-B therapy and received other chemotherapy because of sustained neutropenia, and one patient (2%) died of sepsis during therapy. The factors that adversely affected the CR rate were by stage IV, the presence of B symptoms, the presence of a large mass (greater than 5 cm), and low serum total protein level. The 4-year survival for all 49 patients was 70% and the 4-year disease-free survival (DFS) for the 40 CR patients was 77%. Relapses were higher in patients whose initial serum lactic dehydrogenase (LDH) level was higher than 660 IU/1 (DSF 89% vs. 49%). Toxicity was substantial but acceptable, with neutropenia and mucositis proving to be the most frequent severe side-effects. These preliminary results confirmed the effectiveness of MACOP-B therapy for diffuse non-Hodgkin's lymphoma.

Expression of a multidrug-resistance gene in human malignant lymphoma and related disorders.

The expression of mdr1 gene was measured to determine whether it plays a role in clinical resistance to chemotherapy of human malignant lymphomas. mdr1 expression was found in 4 of 9 cases resistant to chemotherapy. Expression of mdr1 was not detectable in any of 7 chemotherapy-sensitive tumors. The 2 cases of reactive lymphadenitis and the 3 samples of normal mononuclear cells did not show any expression of mdr1 gene, either. These results indicate that expression of the mdr1 gene is not always detectable in cases of malignant lymphoma resistant to chemotherapy, but the detectable expression of mdr1 gene may predict clinical resistance to chemotherapy.