Alternative splicing of exon 14 determines nuclear or cytoplasmic localisation of fmr1 protein isoforms

Impaired expression of the FMR1 gene is responsible for the fragile X mental retardation syndrome. The FMR1 gene encodes a cytoplasmic protein with RNA-binding properties. Its complex alternative splicing leads to several isoforms, whose abundance and specific functions in the cell are not known. We have cloned in expression vectors, cDNAs corresponding to several isoforms. Western blot comparison of the pattern of endogenous FMR1 proteins with these transfected isoforms allowed the tentative identification of the major endogenous isoform as ISO 7 and of a minor band as an isoform lacking exon 14 sequences (ISO 6 or ISO 12), while some other isoforms (ISO 4, ISO 5) were not expressed at detectable levels. Surprisingly, in immunofluorescence studies, the transfected splice variants that exclude exon 14 sequences (and have alternate C-terminal regions) were shown to be nuclear. Such differential localisation was however not seen in subcellular fractionation studies. Analysis of various deletion mutants suggests the presence of a cytoplasmic retention domain encoded in exon 14 and of a nuclear association domain encoded within the first eight exons that appear however to lack a typical nuclear localisation signal.      

液基细胞学结合阴道镜检查在北京市社区妇女宫颈病变筛查中的意义

目的探讨宫颈液基细胞学及阴道镜检查在北京市社区妇女宫颈病变筛查中的临床意义。方法2006年6月至2007年6月对北京市展览路社区的795位20～54岁有性生活的妇女进行筛查。筛查对象接受妇科检查时,留取宫颈超柏氏薄层液基细胞学检测标本,并对宫颈细胞学异常者行阴道镜检查及活组织检查。结果宫颈细胞学阳性[≥ASC-US（不能明确意义的不典型鳞状细胞）]45例,占5．7％（45／795）。其中ASC-US33例,占73．3％（33／45）;低度鳞状上皮内病变8例;高度鳞状上皮内病变3例;不典型腺细胞1例。细胞学阴性750例,占94．3％（750／795）。宫颈细胞学阳性的45例中,5例拒绝行阴道镜检查,占11．1％（5／45）。在行阴道镜活组织病理检查的40例中,慢性宫颈炎11例（27．5％）;宫颈湿疣14例（35．0％）;宫颈上皮内瘤样病变（CIN）1为7例（17．5％）;CIN2为3例（7．5％）;CIN3为4例（10．0％）;早期浸润癌1例（2．5％）。细胞学阴性的750例中,宫颈湿疣2例（0．3％）;CIN1为5例（0．7％）;宫颈低级别腺上皮内病变1例（0．1％）。宫颈液基细胞学筛查CIN1及以上宫颈病变和宫颈癌的敏感度71．4％,特异度94．2％,阳性预测值37．5％,阴性预测值99．2％;筛查CIN2及以上宫颈病变和宫颈癌的敏感度100．0％,特异度96．0％,阳性预测值20．5％,阴性预测值100．0％。结论应重视并及时进行北京市社区人群宫颈病变的早期筛查,薄层液基细胞学结合阴道镜活组织检查及病理学检查,对提高早期宫颈癌筛查的准确性效果明显。     

锌酞菁脂质体光动力作用引起小鼠肿瘤的细胞程序性死亡

电镜观察了锌酞菁脂质体光动力作用引起小鼠ＭＳ－２纤维肉瘤的形态学变化。发现其作用很强，并对肿瘤细胞有明显的直接影响。肿瘤细胞的结构表现出明显的程序性细胞死亡（ａｐｏｐｔｏｓｉｓ，ｐｒｏｇｒａｍｍｅｄｃｅｌｄｅａｔｈ）的特点：胞核染色质凝聚边集、核固缩、核破裂、染色质凝块流失、胞质内吞噬现象、胞膜表面肿胀粗钝的胞突形成、细胞碎裂等。加深了对锌酞菁脂质体光敏作用机理的认识，但其详细的发生机制和调节途径有待阐明。     

Human acute leukemia cell line with the t(4;11) chromosomal rearrangement exhibits B lineage and monocytic characteristics

A cell line, designated RS4;11, was established from the bone marrow of a patient in relapse with an acute leukemia that was characterized by the t(4;11) chromosomal abnormality. The cell line and the patient's fresh leukemic cells both had the t(4;11)(q21;q23) and an isochromosome for the long arm of No. 7. Morphologically, all cells were lymphoid in appearance. Ultrastructurally and cytochemically, approximately 30% of the cells possessed myeloid features. The cells were strongly positive for terminal deoxynucleotidyl transferase. They were HLA-DR positive and expressed surface antigens characteristic for B lineage cells, including those detected by anti-B4, BA-1, BA-2, and PI153/3. Immunoglobulin gene analysis revealed rearrangements of the heavy chain and kappa chain genes. The cells lacked the common acute lymphoblastic leukemia antigen and antigenic markers characteristic of T lineage cells. The cells reacted with the myeloid antibody 1G10 but not with other myeloid monoclonal antibodies. Treatment with 12-O-tetradecanoyl- phorbol-13-acetate induced a monocyte-like phenotype demonstrated by cytochemical, functional, immunologic, and electron microscopic studies. The expression of markers of both early lymphoid and early myeloid cells represents an unusual phenotype and suggests that RS4;11 represents a cell with dual lineage capabilities. To our knowledge, RS4;11 is the first cell line established from t(4;11)-associated acute leukemia.     

Constitutive production of leukemia differentiation, colony- stimulating, erythroid burst-promoting, and pluripoietic factors by a human hepatoma cell line: characterization of the leukemia differentiation factor

Conditioned medium (CM) obtained from a human hepatoma cell line, SK- HEP-1, contains colony-stimulating factors (CSFs) active on murine and human bone marrow-derived granulocyte and macrophage colony-forming units (CFU-GM) and a factor capable of inducing granulocyte-macrophage differentiation (GM-DF) of murine myelomonocytic leukemic cells WEHI- 3B(D+) and human promyelocytic leukemic cells HL-60 when assayed in semisolid agar cultures. The human active granulocyte-macrophage colony- stimulating factor (GM-CSF) for day 7 CFU-GM and the GM-DF for WEHI- 3B(D+) and for HL-60 are not separable by acrylamide agarose column chromatography, eluting at an apparent molecular weight between 20,000 and 35,000 daltons, or by isoelectric focusing (isoelectric point, pH 5.4). In addition, SK-HEP-1 CM contains erythroid burst-promoting activity (BPA) and a factor that promotes the growth of human mixed colonies. SK-HEP-1 cells, which grow as an adherent monolayer, appear not to be endothelial or monocytic in origin since by immunofluorescent staining they are negative for Ia (HLA-DR), monocyte antigen 1 and 2, lysozyme, and factor VIII-related antigen. Positive immunofluorescent staining for keratin and fibronectin suggests the possibility that SK- HEP-1 is an epithelial cell line. Constitutive production of GM-DF as well as other hematopoietic activities including GM-CSF, erythroid BPA, and an activity that promotes the growth of human mixed colony progenitors by a human epithelial tumor cell line, SK-HEP-1, suggests that this cell line is a valuable resource for both large-scale production of these factors and the cloning of the gene(s) that code for these regulators.     

Combination of quinine as a potential reversing agent with mitoxantrone and cytarabine for the treatment of acute leukemias: a randomized multicenter study

A phase III prospective randomized multicenter study was performed to determine whether quinine could improve the response rate of poor-risk acute leukemias (ALs) to standard chemotherapy including a multidrug resistance (MDR)-related cytotoxic agent. The rationale of the study was based on the negative prognostic value of MDR phenotype in ALs and the ability of quinine to reverse this phenotype both in vitro and ex vivo. Three hundred fifteen patients (median age, 49 years; range, 16 to 65) with relapsed (n = 108) or refractory (n = 32) acute myeloblastic leukemia (AML), relapsed (n = 27) or refractory (n = 9) acute lymphoblastic leukemia (ALL), secondary AL (n = 22) or blastic transformation of myelodysplastic syndrome ([MDS] n = 74) or myeloproliferative syndrome ([MPS] n = 43) were randomly assigned to receive mitoxantrone ([MXN] 12 mg/m2/d, days 2 to 5) and cytarabine ([Ara-C] 1 g/m2/12 h, days 1 to 5) alone or in combination with quinine (30 mg/kg/d, days 1 to 5; continuous intravenous infusion beginning 24 hours before MXN infusion). Side effects of quinine were observed in 56 of 161 quinine-treated patients and disappeared in all but four cases after one or two 20% dose decreases. Sera from quinine-treated patients showed increased MXN uptake in an MDR-positive cell line compared with matched sera obtained before quinine infusion. Quinine induced a significant increase in the incidence of nausea, vomiting, mucositis, and cardiac toxicity. A complete response (CR) was observed in 85 of 161 patients (52.8%) from the quinine-treated group versus 70 of 154 patients (45.5%) in the control group (P = .19). The most important differences between quinine and control group CR rates were observed in patients with refractory AMLs and blastic transformation of MDS and MPS. The CR rate was higher in P-glycoprotein-positive cases, although the difference was not significant. Failure of the regimen due to blastic persistence or blast number increase was higher in the control group (61 of 154 patients) than in the quinine group (45 of 161, P = .04). Early death was observed in eight cases (four in each arm) and death in aplasia in 27 cases (20 in quinine group v seven in control group, P = .01). The significant increase of toxicity in the quinine arm could have masked the clinical benefit of MDR reversion in poor- risk ALs.     

Autologous bone marrow transplantation for acute myeloid leukemia using busulfan plus etoposide as a preparative regimen

We have studied the use of a new preparative regimen for the treatment of patients in remission of acute myeloid leukemia (AML) with autologous bone marrow transplantation. Chemotherapy consisted of busulfan 1 mg/kg every 6 hours for 4 days (total dose, 16 mg/kg) on days -7 through -4 followed by an intravenous infusion over 6 to 10 hours of etoposide 60 mg/kg on day -3. Autologous bone marrow, treated in vitro with 100 micrograms/mL of 4-hydroperoxycyclophosphamide, was infused on day 0. We have treated 58 patients up to the age of 60 years, 32 in first remission, 21 in second or third remission, and 5 with primary refractory AML unresponsive to high-dose Ara-C, but achieving remission with aggressive salvage regimens. Of the first remission patients, there has been 1 treatment related death and 5 relapses. With median follow-up of 22 months, the actuarial relapse rate is 22% +/- 9% and disease-free survival is 76% +/- 9% at 3 years. Patients with favorable French-American-British (FAB) subtypes (M3 or M4 EO) did especially well, with no relapses seen in 15 patients observed for a median of 30 months. Actuarial relapse rate at 3 years was 48% for first remission patients with less favorable FAB subtypes. Of patients in second or third remission, there were 5 treatment related deaths and 4 relapses. With median follow-up of 22 months, the actuarial relapse rate is 25% +/- 11% and disease-free survival is 56% +/- 11% at 3 years. Four of five primary refractory patients died during treatment and 1 remains in remission with short follow-up. These preliminary data are very encouraging and, if confirmed, support the use of autologous purged bone marrow transplantation using aggressive preparative regimens as one approach to improve the outcome of adults with AML.