A molecular phylogeny shows the single origin of the Pyrenean subterranean Trechini ground beetles (Coleoptera: Carabidae).

Trechini ground beetles include some of the most spectacular radiations of cave and endogean Coleoptera, but the origin of the subterranean taxa and their typical morphological adaptations (loss of eyes and wings, depigmentation, elongation of body and appendages) have never been studied in a formal phylogenetic framework. We provide here a molecular phylogeny of the Pyrenean subterranean Trechini based on a combination of mitochondrial (cox1, cyb, rrnL, tRNA-Leu, nad1) and nuclear (SSU, LSU) markers of 102 specimens of 90 species. We found all Pyrenean highly modified subterranean taxa to be monophyletic, to the exclusion of all epigean and all subterranean species from other geographical areas (Cantabrian and Iberian mountains, Alps). Within the Pyrenean subterranean clade the three genera (Geotrechus, Aphaenops and Hydraphaenops) were polyphyletic, indicating multiple origins of their special adaptations to different ways of life (endogean, troglobitic or living in deep fissures). Diversification followed a geographical pattern, with two main clades in the western and central-eastern Pyrenees respectively, and several smaller lineages of more restricted range. Based on a Bayesian relaxed-clock approach, and using as an approximation a standard mitochondrial mutation rate of 2.3% MY, we estimate the origin of the subterranean clade at ca. 10 MY. Cladogenetic events in the Pliocene and Pleistocene were almost exclusively within the same geographical area and involving species of the same morphological type.

SIAH-1 inhibits cell growth by altering the mitotic process.

SIAH-1, the human homologue of the drosophila seven in absentia gene, is a p53-p21Waf-1 inducible gene. We report that stable transfection with SIAH-1 of the epithelial breast cancer cell line MCF-7 blocks its growth process. The transfectants show a redistribution of SIAH-1 protein within the nucleus, more specifically to the nuclear matrix, associated to dramatic changes in cell morphology and defective mitosis. Multinucleated giant cells (2-12 nuclei in more than 50% cells) were a most striking observation associated with tubulin spindle disorganization and defective cytokinesis. There were also present at high frequency abortive mitotic figures, DNA bridges and persistance of intercellular bridges and midbodies, along with an increased expression of p21Waf-1. These results indicate that the mechanism of growth arrest induced by SIAH-1 in MCF-7 cells involves disorganization of the mitotic program, mainly during nuclei separation and cytokinesis.

p21WAF-1 reorganizes the nucleus in tumor suppression.

Interphasic nuclear organization has a key function in genome biology. We demonstrate that p21WAF-1, by influencing gene expression and inducing chromosomal repositioning in tumor suppression, plays a major role as a nuclear organizer. Transfection of U937 tumor cells with p21WAF-1 resulted in expression of the HUMSIAH (human seven in absentia homologue), Rb, and Rbr-2 genes and strong suppression of the malignant phenotype. p21(WAF-1) drastically modified the compartmentalization of the nuclear genome. DNase I genome exposure and fluorescence in situ hybridization show, respectively, a displacement of the sensitive sites to the periphery of the nucleus and repositioning of chromosomes 13, 16, 17, and 21. These findings, addressing nuclear architecture modulations, provide potentially significant perspectives for the understanding of tumor suppression.

p53 mutations and overexpression in locally advanced breast cancers.

Alterations in the p53 gene were analysed in 39 patients with locally advanced breast cancers (LABCs) (stage III-IV) with inflammatory signs in most cases (UICC stage T4d = 32 patients) by molecular and immunohistochemical (IHC) approaches. All patients were included in the same therapy protocol. Using polymerase chain reaction (PCR) and a single-strand conformational polymorphism migration technique (SSCP), the presence of mutations in exons 2-11, covering the entire coding sequence of the p53 gene, was evaluated. Using the mouse specific anti-human p53 monoclonal antibody (PAb 1801), we also looked for overexpression of the p53 protein in tissue sections. In 16 cases shifted bands were reproducibly identified by PCR-SSCP, and all but one (localised to exon 10) were in exons 5-8, the usual mutational hotspots. Fifteen of these 16 samples were sequenced and 14 of the suspected mutations (36%) were confirmed. Most of them (12) were single nucleotide substitutions, and transitions were more frequent (eight cases) than transversions (four cases). Fourteen of the tumour samples were positively stained with the monoclonal antibody PAb 1801, 11 with nuclear staining only, two with mixed cytoplasmic and nuclear staining and one with cytoplasmic staining only. Staining patterns were very heterogeneous in terms of the percentage of positive cells (10-75%) and their distribution in the tissue section (isolated foci or dispersed cells). In 11 of the 14 mutated cases a positive immunostaining was observed. The presence of a p53 mutation was significantly associated with larger tumour diameter (chi 2 = 7.490, P = 0.0062) and the presence of clinical metastases (stage IV) (chi 2 = 10.113, P = 0.0015). A non-statistically significant trend of association was observed between p53 mutation, negative oestrogen receptors and lower response rate to therapy. Our results in this group of patients and the heterogeneity of the staining of tumour cells in tissue sections suggest that p53 mutations could be a late event in this non-familial form of breast cancer.

Single-steep transformation of human breast epithelial cells by SV40 large T oncogene.

Normal human mammary epithelial cell (HMEC) cultures originating from 2 mammoplasty reduction surgical samples were transfected with replication-defective SV 40 DNA. Two independent cell lines designated as S2T2 and S1T3, selected for their increased proliferation potential and lifespan, were propagated for greater than 22 months in culture. They maintained a near-diploid karyotype with few chromosomal markers such as trisomy 1q (S1T3) and trisomy 8q (S2T2), which are most common in breast cancer in vivo. Immortalized S1T3 cells were not tumorigenic, whereas S2T2 cells produced slowly growing tumors in nude mice. One tumor was propagated in vitro and the transformed NS2T2 cell line subsequently raised 100% large tumors in the nude mouse. Rearrangement of the SV40 genome was observed in NS2T2 cells, which was not associated with increased expression of large T antigen. S1T3, S2T2 and transformed NS2T2 cell lines expressed cytokeratins CK18, CK19, the mammary-specific antigen DF3, and functional EGF receptors. Single-step immortalization and malignant transformation of human breast epithelial cells can thus occur upon transfection with SV40 large T oncogene. The chromosomal abnormalities observed in these cell lines suggest that they could offer a model for the study of breast-tumor progression in vitro.

Immortalization of human adult normal prostatic epithelial cells by liposomes containing large T-SV40 gene.

Simian virus SV40 has been widely used to immortalize epithelial cells of mammalian origin. We report here, for the first time to our knowledge, the immortalization of normal adult prostatic epithelial cells in culture by transfection of a plasmid containing SV40 genome with a defective replication origin (SV40 ori-) encapsulated into liposomes. These cells (PNT1) have now been cultured for more than 12 months, and shown to contain the SV40 genome. They express large T protein, present the phenotype of differentiated luminal prostatic cells (positive with antibodies to cytokeratin 18, 19, weakly positive for prostatic acid phosphatase and prostatic specific antigen, negative with anticytokeratin 14 and KL2 antibody). PNT1 cells contain high affinity receptors for dihydrotestosterone. These cells provide a useful tool to study the biology and the pathology of adult prostatic epithelial cells, specially to understand the steps leading to prostatic transformation.

Effect of gamma interferon on HLA class-I and -II transcription and protein expression in human breast adenocarcinoma cell lines.

The spontaneous expression of HLA class-I and class-II molecules in 5 human breast carcinoma cell lines, MCF-7, T47D, ZR75-1, HSL-53, MDA-MB 231, and their modulation during IFN-gamma treatment, are reported. The expression of cell-surface determinants was examined by indirect immunofluorescence using monoclonal antibodies (MAbs) specific for HLA class-I and class-II (DR, DQ and DP) antigens. The biosynthesis and maturation of these molecules were analyzed by 2-dimensional gel electrophoresis analysis (2D-PAGE) of class I, DR alpha, beta and invariant immunoprecipitates. Transcription was analyzed by Northern blot hybridization with HLA class-I and -II cDNA-specific probes. In all cell lines, more than 80% of cells expressed HLA class-I antigens at their surface. 2D-PAGE and mRNA studies showed a variable basal level of HLA class-I biosynthesis and transcription with a constant increase after 1,000 U/ml IFN-gamma treatment. HLA class-II determinants were totally absent from the surface of MCF-7, MDA MB231, ZR75-1 and T47D but they were detected in a small subpopulation of HSL-53 cells (DR 6%, DQ 6%, DP 20%). Spontaneous biosynthesis of HLA-DR molecules in immunoprecipitates analyzed by 2D-PAGE or transcripts in Northern blot were not detected in the 5 cell lines. Treatment with 1000 U/ml IFN-gamma induced or increased the expression of HLA class-II molecules in all cell lines but DQ expression was variable. While T47D, ZR75-1 and HSL-53 increased their transcripts and antigen expression, MDA, MB231 and MCF-7 showed no DQ mRNA transcript. Biochemical analysis of the DR products revealed a classical alpha, beta and invariant (li) chain pattern, but indicated a constant glycosylation defect in the invariant chain in all cell lines, associated with weak expression of the beta chain and the presence of an extra spot of low molecular weight in the acidic part of the gel. Thus, post-transcriptional events did not appear to be totally controlled by IFN-gamma in the different cell lines. These differences in DQ expression and glycosylation process in different breast cancer cells may be important in the activation of the immune response among different individuals.

Recombinant gamma interferon provokes resistance of human breast cancer cells to spontaneous and IL-2 activated non-MHC restricted cytotoxicity.

Natural and lymphokine activated killer cells (NK and LAK) are believed to play an important role in the control of tumour progression and metastasis. Their specific receptors on tumours cells are still unknown. Several studies suggest that these cells recognise and eliminate abnormal cells with deleted or reduced expression of MHC class I molecules. Previous reports suggest that interferons (IFN), by increasing MHC class I expression on target cells, induce resistance to killing by NK cells. We investigated the role of MHC molecule expression by two human breast cancer cell lines T47D and ZR75-1 in their susceptibility to NK and LAK cells. These two cell lines spontaneously express low levels of HLA class I antigens but no HLA class II molecules. After IFN-gamma treatment they both overexpressed MHC class I and de novo expressed class II molecules as detected by flow cytometry, quantified by a radioimmunoassay and analysed by two-dimensional gel electrophoresis. Opposed to untreated cells these IFN-gamma treated cells were resistant to NK and LAK lysis. Furthermore, preincubation of IFN-gamma treated breast cancer cells with F(ab')2 fragments of monoclonal antibodies to HLA class I and HLA class II molecules was unable to restore lysis. In contrast, several complete monoclonal antibodies including anti-HLA class I and HLA class II induced the lysis of target cells whether or not they had been treated by IFN-gamma. The therapeutic use of monoclonal antibodies directed against antigens expressed on tumour cells (ADCC) in conjunction with interferon therapy should be discussed in lymphokine-based strategies for treatment of cancer patients.

Interleukin-2 after autologous bone marrow transplantation as consolidative immunotherapy against minimal residual disease.

Recipients of autologous BMT demonstrate clinically significant immune deficiency, particularly involving the T lymphocytes. While quantitatively the immune system generally returns to normal during the first 3 months, there is a prolonged delay in the recovery of qualitative immune functions. T cell proliferation is impaired immediately after transplantation and slowly recovers over a period of more than 1 year. In addition, a defect has been documented in IL-2 producing cells and may be of major importance in the pathophysiology of this immunodeficiency. However, post-ABMT, PHA-stimulated T cells are TAC+ and are able to respond to exogenous IL-2 in vitro. Very early after ABMT, NK and LAK activities of PBMC normalize but are significantly increased in vitro by IL-2. On this basis, a clinical assessment of rIL-2 administration on the immunological reconstitution of ABMT patients and as consolidation immunotherapy against minimal disease has been initiated in a phase I/II study.

Genetic thrombocytopenia with an autosomal dominant transmission: a study of 54 cases.

On the basis of a retrospective study of 3,600 platelet kinetic studies, we have isolated 54 cases with chronic thrombocytopenia, a normal autologous and homologous platelet lifespan, and increased mean platelet volume without Döhle bodies, the absence of any functional platelet abnormalities, and a normal megakaryocyte count. These cases were either discovered during the first few years of life (i.e. constitutional) and/or were proven to be familial (autosomal dominant transmission). Previous treatments (corticosteroids, immunoglobulins, androgens, immunosuppressor agents, splenectomy) were not effective in any of these cases or in their relatives. A new syndrome can therefore be proposed which can be easily suspected in cases of early onset in life, increased platelet volume, failure of corticosteroids or evidence of a familial blood disorder. It can be proved when the autologous platelet life span is demonstrated to be normal in spite of a chronic thrombocytopenia and a normal megakaryocytic count. The recognition of this syndrome will avoid neonatal complications (cephal-hematomas), surgical complications, and the use of expensive and possibility harmful ineffective treatments, both in the propositus and in other abnormal family members. The syndrome is certainly frequent (54 families are presented here), but the diagnosis is often missed or delayed due to the low risk of hemorrhage. However, it is associated with a certain risk of leukemia (4 cases in 3 families).

HLA-DR and DQ antigens in chronic lymphocytic leukemia: dissociation of expression revealed by cell surface, protein, and mRNA studies.

We studied peripheral blood lymphocytes (PBL) of eight B chronic lymphocytic leukemia (CLL) patients for the expression of the human leucocyte antigens, HLA-DR and HLA-DQ. Cell surface expression of HLA class II epitopes was analyzed by fluorescent activated cell sorter (FACS) using three monomorphic anti-HLA class II monoclonal antibodies (mAb) specific for DR (D1.12) and DQ (TU22, L2) and a polymorphic anti-DQ (G2A5). The DR and DQ molecules were characterized by two-dimensional gel electrophoresis (2D-PAGE) of the specific immunoprecipitates from biosynthetically labeled cells. DR, DQ specific probes were used to characterize the class II transcripts of the corresponding genes. The data obtained with immunofluorescence disclosed two distinct patterns of HLA class II expression leading to two cell surface phenotypes: (DR+DQ+) and (DR+DQ-). In all cases the cells expressed normal amounts of HLA-DR gene products in terms of mRNA. DR cell surface determinants were present in more than 80% of cells in every sample. By 2-D gel analysis DR proteins disclosed the normal classical pattern associating the alpha, beta, and invariant chain gamma with normal level of biosynthesis for alpha and gamma but decreased biosynthesis for one of the beta gene products. Moreover, the three chains demonstrated defect in glycosylation process. In half of the cases studied the cells lacked DQ molecules at the cell surface. DQ alpha and DQ beta transcripts were detected in all cases, although the amount was extremely low in one case. DQ proteins were variable in the DQ+ phenotype and absent in the DQ- one. Interestingly, TPA and rIFN gamma treatment could restore normal glycosylation process of the DR isotype and increase biosynthesis of DQ alpha and beta chains. Those combined results support the view that transcriptional, post-transcriptional, and posttranslational mechanisms underlie the heterogeneity of class II expression observed in CLL. Moreover, 2-D gel analysis may be an invaluable tool for the analysis of the biosynthetic process of class II molecules.

Quantitative modifications of major histocompatibility complex (MHC) antigens induced by recombinant gamma interferon in two human breast cancer lines.

H466-B and T47-D breast carcinoma cell lines were treated with recombinant gamma interferon (r gamma IFN) to study major histocompatibility complex (MHC) class I and class II antigen responses. Untreated H466-B cells released B2 microglobulin (B2M) into the culture medium and expressed B2M and class I heavy chain on 100% of the cells. The expression of class II antigens (DR) was limited to 8 +/- 4% of the cells. This subpopulation was isolated by cell sorting and labelled with 35S methionine. Protein extracts were immunoprecipitated with anti-DR antibody and subjected to two dimensional non-equilibrium pH gradient electrophoresis (2D-PAGE). A normal pattern of expression of invariant, alpha and beta chains was shown. The MHC antigenic expression of H466-B parental cell line was not modified by interferon treatment. Untreated T47-D cells did not release B2M into the culture medium, expressed B2M and class I heavy chain on 100% of the cells but did not express class II molecules using radio-immunoassay or 2D-PAGE. As early as 24 h after r gamma IFN addition, T47-D cells released B2M into the medium, B2M and class I heavy chain were significantly greater than that of untreated cells, and class II antigenic expression was found, all these in a dose dependent manner. 2D-PAGE analysis of class II antigens revealed the profile of human DR molecules but this expression seemed incomplete since only single alpha and beta spots were detected suggesting a possible defect in the sialilation of DR molecules. These results show a heterogeneity in MHC antigenic responses to r gamma IFN and suggest that synthetized class II molecules may be incompletely processed.

Changes in the patterns of protein phosphorylation associated with granulocytic and monocytic-induced differentiation of HL-60 cells.

Using two-dimensional gel electrophoresis we have analyzed the pattern of phosphorylated proteins in HL-60 leukemia cells and changes associated with their differentiation into granulocyte and monocyte-like cells. In undifferentiated cells 18 spots with MW ranging from 110 to 17, kDa were individualized with high resolution and reproducibility. Myelocytic differentiation induced by dimethyl sulfoxide (DMSO) and retinoic acid (RA) resulted in a decrease of the overall phosphorylation, the disappearance of two proteins of 42 and 17 kDa, and the appearance of one new acidic protein of 46-48 kDa. These changes seem to be specifically related to this differentiation pathway since they are not found in two HL-60 subclones resistant to DMSO or RA-induced differentiation. Monocytic differentiation induced by 1,25-dihydroxyvitamin D3 [1,25 (OH)2D3] and the combination of RA + 1-B-D-arabinofuranosyl cytosine (Ara-C) was associated with the appearance of 2 proteins of 68 kDa and 2 proteins of 80 kDa located in the acidic region of the gel. The protein of 17 kDa, when disappeared completely in granulocytic-like cells was present in monocytic cells, this suggesting that its phosphorylation state may be involved in the control of the differentiation pathway of HL-60 cells. Data concerning the effect of phorbol myristate acetate (PMA) and histamine on the level of phosphorylation of various proteins in HL-60 cells have also been obtained and discussed. Our results show that the myelocytic and monocytic phenotypes are characterized by a specific pattern of phosphoproteins involving both phosphorylation and dephosphorylation reactions.

Differential expression of HLA-DR and HLA-DC/DS molecules in a patient with hairy cell leukemia: restoration of HLA-DC/DS expression by (12-0-tetradecanoyl phorbol-13-acetate), 5 azacytidine, and sodium butyrate.

Biosynthesis and molecular structure of major histocompatibility complex (MHC) class II antigens of DR2/DR7 hairy cells were analyzed by two-dimensional polyacrylamide-gel electrophoresis (2D-PAGE). Two anti-human Ia monoclonal antibodies (mAb) were used to immunoprecipitate DR and DR-linked DC/DS molecules. Monoclonal antibody VI 15 C recognizes DR (I-E-like) molecules and CA 2.06 precipitates DR and DR-linked DC/DS (I-A-like) molecules in DR7 allotypes. Studies were performed on a pure population of hairy cells before and after culture with phorbol ester: 12-O-tetradecanoyl phorbol-13-acetate (TPA), 5 azacytidine (5 Aza), sodium butyrate (NA-BU), and phytohemagglutinin (PHA-P). Before any treatment, hairy cells expressed and synthesized DR antigens: DR alpha and beta subunits appeared both qualitatively and quantitatively normal by 2D-PAGE profile. In contrast, the hairy cells failed to express and synthesized any DC/DS molecule. The lack of DC/DS molecular expression was restored after culture in presence of TPA, sodium butyrate, and 5 azacytidine, but not after PHA-P treatment. Differential molecular expression of MHC class II antigens in leukemic cells provides a model to define further discrete stages of hemopoietic differentiation and study the role of these molecules in the cellular interactions occurring during differentiation.

In vitro bone marrow GM-CFC growth in aplastic patients. Correlation between E rosette forming cells and immunotherapy results.

In vitro bone marrow GM-CFC (granulomonocyte colony forming cell) growth following E rosette (+) cell depletion was studied in 14 aplastic patients to determine whether an autoimmune factor could be involved in the pathophysiological process leading to the decrease in bone marrow colony formation. The increase in GM-CFC growth after E rosette (+) cell depletion was high in 8 cases, suggesting that in these cases an autoimmune mechanism may have been involved. All these patients responded within a month to treatment with antithymocyte globulin (ATG) or corticosteroids. Six patients did not respond to immunotherapy, none showed increased GM-CFC growth. The increase of in vitro GM-CFC growth after E rosette (+) cell depletion in aplastic anemia could therefore be a good indication for the trial of immunotherapy. However this study seems to be useful only in patients with at least a few months evolution after diagnosis.

Inhibitory effects of peripheral blood cells on in vitro colony formation by autologous bone marrow in aplastic anaemia: relation with response to immunosuppressive therapy.

The inhibitory activity of peripheral blood lymphocytes on autologous bone marrow was studied in 27 patients with aplastic anaemia after treatment with androgen. Inhibitory activity was hard to assess in 10 patients studied during the first year of treatment. The colony count was too low to be certain of differences between the samples incubated with or without lymphocytes. Among the 17 patients who had more than 10 colonies per 2 x 10(5) mononuclear bone marrow cells, nine showed inhibitory activity by peripheral blood lymphocytes. After 12 months of androgen therapy each of these patients showing inhibitory activity of bone marrow colony forming cells by peripheral lymphocytes responded to antithymocyte globulin. None of nine patients with few colony forming cells or no inhibitory activity of lymphocytes responded to immunosuppression.

Syndrome of neutrophil agranulocytosis, hypogammaglobulinemia, and thymoma.

The clinical, hematologic, and immunologic findings of a syndrome of agranulocytosis, hypogammaglobulinemia, and thymoma are described. Neutrophil agranulocytosis predisposing to severe infectious disease resulted from a deficiency of mature cells in bone marrow. Autologous and heterologous stem cell growth in vitro was inhibited by the patient's serum. Immunoglobulin deficiency was secondary to the absence of peripheral blood B lymphocytes, while T-cell subpopulations and cellular immunity were present. Surgical removal of a spindle cell thymoma had no effect on the agranulocytosis and B-cell deficiency. The hematologic findings did not respond to steroid therapy and cyclophosphamide. However, the agranulocytosis improved with repeated plasmapheresis and the patient achieved a clinical remission.

[Mechanisms and prognosis of neutropenia in Felty's syndrome. 27 cases (author's transl)]. / Mécanismes et pronostic de la neutropénie du syndrome de Felty. 27 observations.

Twenty patients with Felty's syndrome were investigated. Isotopic studies of polymorphonuclear neutrophils, bone marrow biopsies and autoradiographies, and cultures of granulous stem cells showed that neutropenia resulted from three mechanisms acting simultaneously: hypermargination of the neutrophils predominantly in the spleen, decreased production of granulocytes in the bone marrow, and peripheral hyperdestruction of the neutrophils. Anti-granulocyte antibodies were detected in 3/12 patients. Other factors present in the serum of 2/4 patients seem capable of inhibiting the growth of granulocytic stem cells. Secondary bacterial infection (77%) may explain the severity of the prognosis: 13 out of 27 patients died 4 years on average after neutropenia was diagnosed.