Inhibition of presenilin 1 expression is promoted by p53 and p21WAF-1 and results in apoptosis and tumor suppression.

Previously, we cloned a cDNA fragment, TSIP 2 (tumor suppressor inhibited pathway clone 2), that detects by northern blot analysis of M1-LTR6 cells a 3-kb mRNA downregulated during p53-induced apoptosis. Cloning the full-length TSIP 2 cDNA showed that it corresponds to the presenilin 1 (PS1) gene, in which mutations have been reported in early-onset familial Alzheimer's disease. Here we demonstrate that PS1 is downregulated in a series of model systems for p53-dependent and p53-independent apoptosis and tumor suppression. To investigate the biological relevance of this downregulation, we stably transfected U937 cells with antisense PS1 cDNA. The downregulation of PS1 in these U937 transfectants results in reduced growth with an increased fraction of the cells in apoptosis. When injected into mice homozygous for severe combined immunodeficiency disease (scid/scid mice), these cells show a suppression of their malignant phenotype. Our results indicate that PS1, initially identified in a neurodegenerative disease, may also be involved in the regulation of cancer-related pathways.

Expression of a public idiotype by human monoclonal IgM directed to myelin-associated glycoprotein and characterization of the variability subgroup of their heavy and light chains.

Most studies using rabbit or mouse antisera failed to detect CRI between human IgM directed to MAG. We show here that 9 of 10 such IgM express a public CRI as defined by a nonhuman primate antiserum. Shared idiotype is likely involved in (or close to) the combining site of those IgM since antiidiotypic serum inhibited the binding of IgM to MAG and reacted with IgM having different variable regions of light and heavy chains. Partial aminoterminal sequence of heavy and light chains showed that anti-MAG IgM use either lambda chains (one IgM) or kappa light chains (six IgM) of different variability subgroups (V kappa IV in three instances, V kappa I in two, and V kappa II in one), whereas heavy chains belong to the VHIII (six IgM) or to the VHII (1 IgM) subgroup. These features distinguish these IgM from other human monoclonal IgM with a defined antibody activity, such as rheumatoid factors or cold agglutinins.

Immunohistological study of precancerous mucus modification in human distal colonic polyps.

The M1 antigens associated with gastric fucomucins, oncofetal markers of the distal colonic mucosa, were demonstrated to be more closely associated with adenomas [92 of 139 (66%)] than with invasive adenocarcinomas [27 of 218 (12%)]. They were always expressed in tumors containing the M3 antigen normally associated with the intestinal mucus. The M1 antigens, present in 100% of hyperplastic polyps (30 of 30), were not specific for a particular histological type of adenoma but were found to be more closely associated with those showing a villous differentiation [41 of 47 (87%)] than with those having a tubular pattern [51 of 92 (55%)]. The presence of these M1 antigens depended neither on the size nor on the degree of cytological atypia of the nodular adenomas. However, M1 antigens were found in 94% of the adenomas (35 of 37) concomitant with adenocarcinomas; in contrast, only 56% of adenomas (55 of 102) observed on noncancerous mucosa contained these M1 antigens. As already demonstrated during rat colonic carcinogenesis, mucus modification characterized by the presence of M1 antigens could represent early molecular changes occurring before malignant transformation related to a chemical carcinogen. These M1 antigens might be regarded as early precancerous markers of an oncofetal type, associated with human distal colonic mucosa.

Ectopic expression of Bcl-2 switches over nuclear signalling for cAMP-induced apoptosis to granulocytic differentiation.

The IPC-81 myeloid leukaemia cells undergo apoptosis rapidly after cAMP stimulation (6 h) and cell death is prevented by early over-expression of the cAMP-inducible transcription repressor ICER, that blocks cAMP-dependent nuclear signalling. Therefore, the expression of specific genes controlled by CRE-containing promoters is likely to determine cell fate. We now show that cAMP-induced cell death also is abrogated by the over-expression of the anti-apoptotic gene, Bcl-2. Contrary to ICER, Bcl-2 does not affect cAMP-signalling and allows the analysis of cAMP responses in death rescued cells. The Bcl-2 transfected cells treated with 8-CPT-cAMP were growth-arrested and thereafter cells embarked in granulocytic differentiation, with no additional stimulation. Neutrophilic polynuclear granulocytes benefited from a long life span in G0-G1 and remained functional (phagocytosis). This work demonstrates that, using anti-apoptosis regulators, 'death signals' could be exploited to trigger distinct biological responses. Indeed, cAMP signal can trigger several simultaneously developing biological programs, in the same cell, i.e., growth regulation, apoptosis and differentiation. This cell system should prove useful to determine how a tumour cell can be re-programmed for either apoptosis or functional maturation by physiological signals.

A new extra sequence at the amino terminal of a mu heavy chain disease protein (DAG).

The primary structure of a human mu heavy chain (DAG) protein is described. The native protein is a circular decamer with a molecular weight (Mr) of 500 kDa, each decamer being constituted of the constant domains C mu 2, C mu 3 and C mu4 and interlinked by 15 disulfide bridges. At its NH2-terminal each monomeric chain starts with an "extra sequence". The amino acid sequence of this segment is Arg-Gln-Ser-Asp-Asp-Pro-Val-Leu-Arg-Gly-Thr-Thr-Val-Pro-Val-Thr-Glu and its reinitiation point is located at Val223 (Gal numbering), at the beginning of C mu 2. This sequence has no homology with any other protein included in the present databases.

Ciliated differentiation of rabbit tracheal epithelial cells in vitro.

Primary cultures of rabbit tracheal epithelial (RbTE) cells have been performed in two different ways. Quantitative analysis of both proliferative capacities and ciliated differentiation process were carried out using epithelial cell cultures from tracheal explants and from dissociated tracheal epithelial cells in air-liquid interface conditions. We show that both alpha- and beta-tubulins from RbTE cells are polyglutamylated and that this posttranslational modification is restricted to cilia axonemes and centrioles of non-ciliated cells. A monoclonal antibody raised against polyglutamylated tubulins was used to quantify the proportion of ciliated cells. Even though epithelial cells from outgrowths obtained by the explant technique highly proliferated during the first days of culture, no ciliated differentiation occurred. On the other hand, using air-liquid interface conditions after proliferation of dissociated cells, we could observe and quantify a ciliated cell differentiation in vitro by both Western blot and flow cytometric analysis. The specific detection and quantification of ciliated cells open the way for the biochemical and molecular characterization of centriolar components during ciliated differentiation.

Immunological comparison of ovarian and colonic CEA.

Ovarian and colonic CEA were compared immunologically by means of antisera prepared against each of them. CEAs of both origins were found identical by immunodiffusion methods. In radioimmunological experiments, slight differences were observed between some but not all ovarian CEAs and colonic CEAs and also between different preparations of colonic CEA: no organ specificity of ovarian CEA could be demonstrated. Finally, CEA level was measured in 41 sera of patients with ovarian carcinoma by two radioimmunoassays, one using colonic CEA as tracer and standard and anti-colonic CEA serum, the other using ovarian CEA and anti-ovarian CEA serum: the values given by the two assays were highly correlated (rs = 0.8107), meaning that an organ specific assay for ovarian CEA is not needed.

Fanconi anemia. Chromosome breakage and cell cycle studies.

The experience with cytogenetic and flow cytometry methods used for the diagnosis of Fanconi anemia (FA) in one center is summarized. The tests consist of chromosomal breakage and cell cycle studied after sensitization by the introduction of nitrogen mustard into phytohemagglutinin-stimulated blood-cell cultures. The cytogenetic test was shown to be reliable in ascertaining the diagnosis of FA. Flow cytometry studies showed a marked increase in the percentage of cells in G2/M phase in FA patients after sensitization by nitrogen mustard. This increase, however, could not be detected in three FA patients with myelodysplasia or acute leukemia and the results were ambiguous on three occasions.

Use of fluorescent probes to assess the early sulfhydryl depletion and oxidative stress induced by mechlorethamine in human bronchial epithelial cells.

In this study, we report in vitro methods using fluorescent probes to assess thiol depletion and the oxidative stress induced by mechlorethamine (HN2), a nitrogen mustard, on a human bronchial epithelial cell line (16HBE14o-). Monobromobimane (mBBr) and 2',7'-dichlorofluorescin-diacetate (H(2)DCF-DA) were respectively used to monitor the intracellular thiol and peroxide levels. Fluorescent measurements were realized on gated live cells with a flow cytometer and a microspectrofluorimeter. Results clearly show that HN2 induced an early reduction of free sulfhydryl groups inside the cell correlated with an increase in the intracellular peroxides content. HN2 effects were time and dose dependent. The use of these fluorescent probes provide a useful and rapid methods for future screening of protective molecules against the early sulfydryl depletion and oxidative stress induced by HN2 on human airway epithelium.

A tumor-associated antigen in human nephroblastomas.

Antisera prepared against extracts of human nephroblastomas (Wilms' tumors) allowed the characterization in these extracts, and in a few extracts of tumors of other organs, of a tumor-associated antigen. This antigen is different from all previously described antigens, and was named W antigen (W for Wilms). It does not seem to be a carcinoembryonic antigen, and hence it could be of viral origin.

[Comparative study of the resistance of CEA and NCA to acid proteases]. / Etude comparative de la résistance aux protéases acides du CEA et du NCA

The non-specific cross reacting antigen (NCA), characterized by its cross reaction with the carcinoembryonic antigen of the digestive system (CEA), is present in human polymorphonuclears and macrophages. We showed that this antigen is not damaged by endocellular acid proteases (cathepsine D and E), nor by pepsin: there is no change in the molecular weight, the electrophoretic mobility and the immunochemical reactivity of the enzyme treated NCA. These data make likely the hypothesis of NCA playing a role in the protection of polymorphonuclears and macrophages against their own proteases.

Protein Rou. A human IgA hybrid.

Protein Rou is a human IgA2 myeloma protein that carries the isoallotype marker n A2m(2). Partial amino acid sequence of its H chain (alpha) shows that the hinge region and the CH2 domain are homologous to alpha 2-chain and the CH1 and the CH3 domains homologous to alpha 1. Moreover, the CH1 domain contains the H-L disulfide bond identical to alpha 1. It is concluded that Rou H chain is a hybrid molecule caused by a recombination between alpha 1 and alpha 2 genes. The recombination event occurred between alpha 1-exon 1 and alpha 2-exon hinge and corresponds to position 222-223 of the alpha-chain.

Monoclonal IgM from patients with peripheral demyelinating neuropathies cross-react with bacterial polypeptides.

Human monoclonal IgM associated with a demyelinating peripheral neuropathy often feature a distinct antibody activity directed against a glucuronyl sulphate epitope shared by myelin-associated glycoprotein (MAG), nerve glycolipids and low molecular weight peripheral nerve polypeptides. Earlier studies showed that these IgM use a diverse repertoire of VH and VL genes which exhibit somatic mutations, possibly indicative of an antigen-driven process. Here, we investigated whether such monoclonal IgM may react with environmental bacterial antigens. We found that six patients' sera and purified monoclonal IgM, as well as IgM from supernatants of three clonal anti-MAG-secreting cell lines reacted with unique 90-100 kD polypeptides from extracts of two out of 10 bacterial species. Purified MAG was able to inhibit this reactivity. These results indicate molecular mimicry as a possible mechanism of this immunomediated neuropathy and associated clonal lymphoid disease.

Modulation of spontaneous B-cell differentiation in macroglobulinemia by retinoic acid.

We previously showed that clonal blood B cells from patients with macroglobulinemia spontaneously differentiate in vitro to plasma cells. This process is dependent on an interleukin (IL)-6 autocrine pathway. We investigate here whether all-trans-retinoic acid (RA) interferes with B-cell differentiation either in patients with IgM gammapathy of undetermined significance (MGUS) or Waldenström's macroglobulinemia (WM). RA at a concentration of 10(-5) to 10(-8) mol/L inhibited by 50% to 80% the in vitro differentiation of purified B cells from four of five patients with MGUS and from one of five patients with WM as assessed by the IgM content of day 7 culture supernatants. We next determined whether this effect could be related to an inhibition of IL-6 secretion by cultured B cells and/or a downregulation of the IL-6 receptor (IL-6R), which was constitutively expressed on patients' blood B cells. A 50% to 100% (mean, 80%) inhibition of IL-6 production was found in seven of 10 patients (five with MGUS and two with WM). The IL-6R was no more detectable on cells from patients with MGUS after 2 days of treatment with RA and slightly downregulated in patients with WM. It was of interest that B cells susceptible to the action of RA belonged mostly to patients with IgM MGUS, which reinforces our previous data showing distinct requirements for IL-6-dependent differentiation of blood B cells from patients with VM or IgM MGUS.

Amount of the two major Ag-NOR proteins, nucleolin, and protein B23 is cell-cycle dependent.

To know the biological basis allowing the use of Ag-NOR protein expression as proliferation marker in human malignancies, the relationship between cell cycle and amount of Ag-NOR protein was analyzed. The quantification of the two major Ag-NOR proteins, nucleolin and protein B23, was performed in exponentially growing, serum-deprived, and cell-cycle stimulated cells. Expression of nucleolin was low in serum-deprived cells and increased mostly in S phase during cell-cycle stimulation. Conversely, expression of protein B23 was slightly repressed in serum-deprived cells, and increased progressively until G2 phase during cell-cycle stimulation. The accumulation of nucleolin and protein B23 in G2 compared to G1 was demonstrated using sorted phase-specific cells. In G0, cells sorted according to their very low RNA content, and the amount of Ag-NOR proteins was half of that found in G1 cells, nucleolin being only weakly detectable. Therefore, the expression of nucleolin increased between G0-G1 and G1-S phases. These data support the hypothesis that quantification of Ag-NOR proteins is an estimation of the percentage of cells in each cell cycle phase because their amount is high in S-G2 and low in G1 phases.

Sinefungin and taxol effects on cell cycle and cytoskeleton of Leishmania donovani promastigotes.

Sinefungin is an antibiotic possessing a strong anti-leishmanial activity. Among the most important effects of this molecule on Leishmania donovani promastigotes are morphological modifications and a very rapid and effective inhibition of DNA synthesis. These cells contain a single DNA-rich mitochondrion whose division cycle is coordinated with the nuclear division cycle. We have developed a flow-cytometric procedure based upon mithramycin as fluorochrome that can perform quantitative cell cycle analysis on the nuclear DNA. Cell cycle progression was analyzed to establish that sinefungin irreversibly blocks the promastigotes in early S phase. Sinefungin did not react with stationary cells as they were arrested in G1. Surprisingly, taxol, a microtubule-stabilizing drug, induced the same morphological modifications as sinefungin although it interfered with the G2/M progression. According to immunofluorescence studies, the stable microtubular network is apparently affected neither by taxol nor by sinefungin.

R-phycoerythrin-cyanine 5 tandem discerns CD72 polymorphism.

By fluorescence analysis, we demonstrated that a fluorochrome tandem composed of R-phycoerythrin and cyanine 5 specifically recognized B cells from SJL, AKR, MRL/Mp, and NOD mouse strains, whereas B cells from C57BL/6, DBA/2, SWR, 129/Sv, and BALB/c were not stained. A strict correlation was observed between the fixation of the fluorochrome and the pattern of expression of the pan-B cell marker CD72, i.e., early expression in B-cell lineage development and downregulation on the terminally differentiated activated B cells. Three allelic forms, CD72a, CD72b and CD72c have been well characterized, and show a high number of amino acid substitutions concentrated in the membrane-distal extracellular domain. Using a PCR approach, we determined that all mouse strains positive for fluorochrome staining display the CD72c allelic form. Moreover, a genetic analysis showed that the fixation of the fluorochrome on the B cell is exclusively dependent on the presence of the CD72c allele. Together, these results strongly suggest that the tandem binds a molecular complex comprising the CD72c molecule or recognizes directly the CD72c molecule itself.

Genomic alterations in a case of alpha heavy chain disease leading to the generation of composite exons from the JH region.

Human alpha heavy chain disease (HCD) is characterized by the presence in patient's serum of a short Ig alpha chain devoid of light chains. We analyzed the serum protein, the alpha HCD mRNA and the productive rearranged H chain gene from the leukemic cells of a new case (YAO) of alpha HCD. The abnormal YAO alpha 1 Ig was devoid of VH and CH1 domains and started at the beginning of the hinge region. The alpha HCD mRNA was shorter than normal alpha mRNA and the cDNA prepared from YAO mRNA encoded a leader sequence, an insert of 70 nucleotides and the CH2 and CH3 exons. The origin of the inserted sequence was assessed by cloning and sequence analysis of the alpha 1 productive gene. It started with a leader exon, a leader-VH intron and the first 11 bp of a VH exon. Then the VH region was deleted and replaced by a 19-nucleotide sequence that turned out to correspond to the 3' part of a modified JH5 exon. It was followed by a 221-bp sequence homologous to the JH5-psi JH3 intron and by an inserted sequence of unknown origin. The 3' part of this insertion and the remnant of a JH6 exon delineated a third exon that was followed by a relatively conserved JH6-C alpha intron. These two composite exons were flanked by splicing sites and accounted for the 70-nucleotide insert of the cDNA. The genomic nucleotide sequence also revealed a large deletion in the switch CH1 region which eliminated normal splicing sites and resulted in splicing of the third exon directly to the CH2 exon.

Phorbol ester-induced promyelocytic leukemia cell adhesion to marrow stromal cells involves fibronectin specific alpha 5 beta 1 integrin receptors.

The human promyelocytic cell line NB4 exhibited a weak adhesion capacity for bone marrow-derived stromal cells and their extracellular matrices (5-15% of adherent cells). Adhesion was enhanced by pulse-treatment of cells with phorbolester (PMA 10(-7) M). Adhesion was induced within minutes, was fibronectin-specific, and affected up to 100% of the treated cells. This biological response to PMA resulted from the activation of protein kinase C (PKC), since PKC inhibitors (staurosporine, sphingosine, CGP 41251, and calphostin C) prevented the phenomenon. Phenotypical analysis of integrin receptor expression (particularly FN receptors VLA-4 and VLA-5) at the membrane of untreated or PMA-treated cells revealed that PMA induced no significant modification of the level of expression of these receptors. However, inhibition studies carried out with anti-VLA monoclonal antibodies demonstrated that the FN-specific adhesion triggered by PKC involved the alpha 5 beta 1 FN-specific receptors (VLA-5). We showed that the binding of NB4 cells to fibronectin was RGD-dependent. PMA-induced adhesion was not correlated to phosphorylation of the VLA-5 receptor. These findings may partially explain the malignant behaviour of these cells: The loss of their capacity to adhere to stromal cells may arrest differentiation and explain the large number of leukemic cells in the circulation.

The level of expression of mu heavy chain modifies the composition of peripheral B cell subpopulations.

The B cell receptor (BCR) has a decisive role in transducing signals required for the development of B cells and their survival in the periphery. However, the processes that initiate these signals remain unclear and concepts of constitutive and ligand-dependent signaling have been proposed. Using a mu-transgenic mouse model, we have analyzed the impact of high surface IgM expression on the composition of the splenic B cell population. kappa-deficient mice homozygous for the H3-mu transgene have B cells with a higher BCR surface density than H3 heterozygous mice. This higher BCR expression is associated with an increase in the percentage and the total number of splenic B cells. In addition, an important proportion of CD23(-)CD21(+) marginal zone (MZ) B cells can be observed in H3 homozygous mice. However, these modifications operate in the absence of impairment of the positive selection process of the H3-mu/lambda1 combination over the H3-mu/lambda2 + 3 ones. These results suggest that (i) a constitutive BCR signaling directly correlated with BCR surface density is responsible for the efficient B cell colonization of the periphery with an accumulation of B cells in the MZ and (ii) a ligand-dependent BCR signal is responsible for the clonotype composition of the mature B cell repertoire.