CDKN1C mutation affecting the PCNA-binding domain as a cause of familial Russell Silver syndrome.

BACKGROUND: Russell Silver syndrome (RSS) leads to prenatal and postnatal growth retardation. About 55% of RSS patients present a loss-of-methylation of the paternal ICR1 domain on chromosome 11p15. CDKN1C is a cell proliferation inhibitor encoded by an imprinted gene in the 11p15 ICR2 domain. CDKN1C mutations lead to Beckwith Wiedemann syndrome (BWS, overgrowth syndrome) and in IMAGe syndrome which associates growth retardation and adrenal insufficiency. We searched for CDKN1C mutations in a cohort of clinically diagnosed RSS patients with no molecular anomaly. METHOD: The coding sequence and intron-exon boundaries of CDKN1C were analysed in 97 RSS patients. The impact of CDKN1C variants on the cell cycle in vitro were determined by flow cytometry. Stability of CDKN1C was studied by western immunoblotting after inhibition of translation with cycloheximide. RESULTS: We identified the novel c.836G>[G;T] (p.Arg279Leu) mutation in a familial case of intrauterine growth retardation (IUGR) with RSS phenotype and no evidence of IMAGe. All the RSS patients inherited this mutation from their mothers (consistent with monoallelic expression from the maternal allele of the gene). A mutation of this amino acid (p.Arg279Pro) has been reported in cases of IMAGe. Functional analysis showed that Arg279Leu (RSS) did not affect the cell cycle, whereas the Arg279Pro mutation (IMAGe) led to a gain of function. Arg279Leu (RSS) led to an increased stability which could explain an increased activity of CDKN1C. CONCLUSIONS: CDKN1C mutations cause dominant maternally transmitted RSS, completing the molecular mirror with BWS. CDKN1C should be investigated in cases with family history of RSS.

Growth-supporting activity of fragment Ba of the human alternative complement pathway for activated murine B lymphocytes.

We have investigated the effects of cleavage of factor B by its activating enzyme, factor D, as well as its activation fragments Bb and Ba, on the growth of mouse spleen B lymphocytes preactivated by LPS. Neither factor B nor factor D show any growth-supporting activity when tested alone. The coaddition of factor B and factor D to serum-free cultures of LPS-preactivated B cell blasts increased the proliferation of the responding cells up to the level obtained by restimulation with LPS. Such growth-supporting activity was shown to be mediated by Ba, whereas Bb did not show any significant effect. Furthermore, this effect was not restricted to the LPS-preactivated B cell blasts; in fact, Ba also supported the growth of in vivo, activated B cell blasts of unprimed mice of the LPS-nonresponder C3H/HeJ strain. In contrast, Ba did not maintain growth of Con A-activated T cells or TCGF-dependent CTL cells. Taken together, these results describe the first biological activity of human Ba as a B cell stimulatory factor.

Impact of p53 expression and microsatellite instability on stage III colon cancer disease-free survival in patients treated by 5-fluorouracil and leucovorin with or without oxaliplatin.

BACKGROUND: The aim was to determine the values of p53 tumour expression and microsatellite instability (MSI) phenotype to predict benefit from adjuvant chemotherapy of colon cancer by 5-fluorouracil and leucovorin (FL) alone or with oxaliplatin (FOLFOX). PATIENTS AND METHODS: This retrospective study included 233 unselected patients with stage III colon cancer treated by FL (n = 124) or FOLFOX (n = 109). The impact of p53 expression and MSI on disease-free survival (DFS) was defined using univariate and multivariate analyses. A Cox proportional hazards model was specifically designed to evaluate the interaction between chemotherapy and these genetic alterations. RESULTS: In univariate analyses, addition of oxaliplatin significantly improved DFS provided that tumour overexpressed p53 [hazard ratio (HR) 0.39; 95% confidence interval (CI) 0.19-0.82; P = 0.01] or displayed MSI phenotype (HR 0.17; 95% CI 0.04-0.68; P = 0.01). In multivariate analyses, p53 was confirmed as an independent factor predictive of benefit from FOLFOX (P = 0.03), while the interaction of MSI with chemotherapy could not be determined in the absence of relapse in the MSI group treated with FOLFOX. CONCLUSION: Our observations indicate that MSI status and p53 expression may influence the impact of oxaliplatin on adjuvant treatment of stage III colon cancer patients.

Frequent alteration of DNA damage signalling and repair pathways in human colorectal cancers with microsatellite instability.

Accumulation of frameshift mutations at genes containing coding mononucleotide repeats is thought to be the major molecular mechanism by which mismatch repair-deficient cells accumulate functional alterations. These mutations resulting from microsatellite instability (MSI) can affect genes involved in pathways with a putative oncogenic role, but may also arise in genes without any expected role in MSI carcinogenesis because of the high mutation background of these tumours. We here screened 39 MSI colorectal tumours for the presence of mutations in 25 genes involved in DNA damage signalling and repair pathways. Using a maximum likelihood statistical method, these genes were divided into two different groups that differed significantly in their mutation frequencies, and likely represent mutations that do or do not provide selective pressure during MSI tumour progression. Interestingly, the so-called real-target mutational events were found to be distributed among genes involved in different functional pathways of the DNA metabolism, for example, DNA damage signalling (DNA-PKcs, ATR), double-strand break (DSB) repair (DNA-PKcs, RAD50), mismatch repair (MSH3, MSH6, MBD4) and replication (POLD3). In particular, mutations in MRE11 and/or RAD50 were observed in the vast majority of the tumours and resulted in the concomitant loss of immunohistochemical expression of both proteins. These data might explain why MSI colorectal cancers (CRC) behave differently in response to a wide variety of chemotherapeutic agents, notably those targeting DNA. More generally, they give further insights into how MSI leads to functional changes with synergistic effects in oncogenic pathways.

Microsatellite instability and mutation analysis of candidate genes in urothelial cell carcinomas of upper urinary tract.

A subset of upper urinary tract urothelial cell carcinomas (UUC), arising sporadically or as a manifestation of hereditary non-polyposis colorectal cancer, displays microsatellite instability (MSI). MSI tumours are characterized by defective mismatch repair and accumulation of frameshift mutations in numerous genes harbouring repeats in their coding sequences. We have evaluated the incidence of MSI in UUC and the intratumoral distribution of mutations in 13 candidate target genes. A total of 58 unselected UUC were screened for MSI using the panel of five mononucleotide markers recently recommended by the National Cancer Institute for a precise MSI assessment. Four tumours displayed MSI (7%), among which at least three had alterations in the genes MSH3, BAX, MRE11, RAD50. Mutations in genes involved in key cellular pathways (ATR, DNA-PKcs, MBD4, TCF-4, MSH6, and BLM) were further detected. BAX and MRE11 mutations tend to present homogeneously within the three MSI UUC. Immunohistochemistry (MLH1, MSH2, MSH6) showed that loss of mismatch repair protein expression occurred in all MSI UUC defining the gene defect and that MRE11 and RAD50 mutations were associated with their concomitant loss expression. In conclusion, MSI UUC represent a small proportion of UUC in which BAX and MRE11 mutations are frequent and may play a role early in UUC tumorigenesis.

The role of the DNA mismatch repair system in the cytotoxicity of the topoisomerase inhibitors camptothecin and etoposide to human colorectal cancer cells.

The DNA mismatch repair (MMR) system is involved in the correction of base/base mismatches and insertion/deletion loops arising during replication. In addition, some of the MMR components participate in recombination and double-strand break repair as well as cell cycle regulation and apoptosis. The inactivation of MMR genes, usually hMSH2 or hMLH1, is associated with human colorectal cancers and is responsible for the characteristic microsatellite instability (MSI)+ phenotype of these tumors. Because MMR is assumed to modulate cytotoxicity to various chemotherapeutic agents that act upon DNA, our objectives have been to define its possible involvement in the cytotoxicity of topoisomerase inhibitors. We have shown that colorectal cancer cell lines defective in DNA MMR exhibit an increased sensitivity to both camptothecin, a topoisomerase I inhibitor, and etoposide, a topoisomerase II inhibitor. Sensitivity to these drugs cannot be predicted by measuring endogenous levels of topoisomerase I and II. Our results also indicate that neither p53 status, nor cell cycle alterations correlate with the sensitivity of colorectal cancer cells to topoisomerase inhibitors. On the other hand, our data showing that resistance to these drugs can be achieved by the functional complementation of hMLH1 in an hMLH1-defective cell line have allowed us to establish that MMR is a critical determinant for chemosensitivity. Interestingly, our observations provide the rationale for the better responsiveness of MSI+ tumors to CPT-11, a camptothecin derivative, which we have observed in patients with metastatic colorectal cancers.

Human glutathione S-transferase M1 null genotype is associated with a high inducibility of cytochrome P450 1A1 gene transcription.

We investigated the transcriptional regulation of cytochrome P450 1A1 (CYP1A1) gene in human lymphoblastoid B cells and report that a high inducibility of CYP1A1 gene transcription by 2,3,7,8-tetrachlorodibenzo-p-dioxin is associated with glutathione S-transferase M1 (GSTM1) null genotype, whereas the presence of at least one GSTM1 allele is correlated with induction of only low levels of CYP1A1 mRNA by 2,3,7,8-tetrachlorodibenzo-p-dioxin. These data underline the major importance of the CYP1A1 inducibility phenotype associated with the homozygous GSTM1 null genotype in chemically induced cancers.

A one-step procedure for preparation of classical pathway (C1q) and alternative pathway (factor D) depleted human serum.

A simple method for preparation of a serum depleted in both C1q and Factor D is described. The hemolytic activities of both pathways are completely abolished and can be fully restored using the respective purified complement components. Furthermore, this serum is useful for studying cell systems since blocking either complement pathway does not require chelating agents.

Stability of microsatellites and minisatellites in Bloom syndrome, a human syndrome of genetic instability.

Bloom syndrome (BS) is a human cancer-prone genetic disorder essentially characterized by a generalized genetic instability including a high level of sister chromatid exchanges (SCEs). Although mutator and hyper-Rec phenotypes of BS cells present analogies with those of bacteria and yeast defective in DNA mismatch repair, we report that (CA)(n) microsatellite alterations are undetectable in BS cells. Thus, our results suggest that the origin of BS mutator phenotype is not a major defect in DNA mismatch repair, allowing us to eliminate an attractive hypothesis for the pleiotropy of BS. We previously suggested that at least some of the intra-allelic rearrangements occurring in minisatellites could result from unequal SCEs. Although SCEs are abnormally frequent in BS cells, the present study failed to show any significant variation of the mutation rates of the two hypermutable minisatellites we analyzed. Thus, our results show that, in spite of an overall genetic instability, alterations in structural motifs known to be predisposed to instability by different mechanisms are undetectable in BS cells.

IgA-affinity purification and characterization of the lectin jacalin.

We describe the use of IgA-Sepharose 4B affinity chromatography to purify the lectin jacalin from saline extracts of Artocarpus integrifolia L. seeds. Elution with 0.8 M D-galactose provides 10-15 mg lectin/50 mg seed protein. Jacalin behaved like a single component on immunoelectrophoresis and a single, somewhat diffuse band was obtained by polyacrylamide gel electrophoresis (PAGE) at pH 4.5. A single peak corresponding to an apparent molecular weight of 43 kDa was obtained by gel filtration on Sephadex G-75 (10 mM phosphate buffered saline (PBS), pH 7.4). On SDS-PAGE +/- 2-mercaptoethanol two bands of apparent molecular weights 11.8 and 14.7 kDa were detected. Jacalin behaved like a protein of apparent molecular weight of 13-14 kDa on Sephadex G-50 eluted with PBS containing 0.2% SDS. These data indicate that the jacalin molecule consists of 3-4 non-identical polypeptide subunits not connected by disulfide bridges. The amino acid composition of IgA affinity-purified jacalin (mol/405 mol amino acids) is Lys (24), His (5), Arg (4), Trp (6), Asx (36), Thr (35), Ser (48), Glx (31), Pro (18), Gly (53), Ala (13), Val (25), Met (3), Ile (23), Leu (25), Tyr (30), Phe (26), which corresponds to a molecular weight of 44.163 kDa.

[Response of metastatic colorectal cancers to treatment with CPT11 (irinotecan): implications of the mismatched base repair system]. / Réponse des cancers colorectaux métastatiques au traitement par CPT11 (irinotécan) : implications du système de réparation des mésappariements de bases.

AIMS: The aim of our study was to assess the potential relationships between tumor responsiveness to CPT11, an analogue of camptothecin, which selectively inhibits DNA topoisomerase I, and the microsatellite instability, a feature of tumors with DNA mismatch repair defect. METHODS: We designed a retrospective clinical study including 35 patients with metastatic colorectal cancer treated with CPT11, for which we analyzed the expression of hMLH1 and hMSH2 in the tumor and determined microsatellite status of repeated mononucleotide tracts present in the coding region of RII-TGFB, BAX, hMSH3 and hMSH6 genes. RESULTS: A partial or minor response was observed in 9 patients, disease stabilization in 14 patients and progression in 12 patients. Staining of hMLH1 was undetectable in 2 of the 35 tumors, while only 1 tumor lacked hMSH2 expression. Four of the 31 tumors analyzed displayed intragenic microsatellite instability. We found a good correlation between inactivation of TGFB-RII, BAX or hMSH3 genes and tumor response to CPT-11 (P =0.002). CONCLUSION: Our preliminary data suggest that intragenic microsatellite instability may influence tumor response to CPT-11 in patients with colorectal cancer.

Involvement of small ArfGAP1 (SMAP1), a novel Arf6-specific GTPase-activating protein, in microsatellite instability oncogenesis.

Small ArfGAP1 (stromal membrane-associated protein 1, SMAP1), a GTPase-activating protein specific for ADP-ribosylation factor 6 (Arf6), which is a small GTPase acting on membrane trafficking and actin remodeling, is frequently mutated in various tumors displaying microsatellite instability (MSI), notably in MSI colorectal cancers (CRC). Genotyping of 93 MSI CRCs (40 stage II, 32 stage III and 21 stage IV) allowed us to underscore that SMAP1 mutation frequency was inversely correlated with disease stage (P=0.01). Analysis of 46 cancer cell lines showed that SMAP1 mutations occurred only in MSI tumors, and consisted exclusively in short insertion or deletion in the coding 10-adenine repeat, generating a premature termination codon located downstream the ArfGAP domain. SMAP1 transcript levels were significant decreased (P=0.006), and truncated SMAP1 protein could not be detected in cells displaying biallelic SMAP1 mutations, owing to its sensitivity to proteasome degradation. To investigate the role of SMAP1 mutations, we used the SMAP1-null HCT116 cell line and we established three isogenic SMAP1-complemented clones. Cell proliferation was first assessed in vivo using subcutaneous xenografts into immunodeficient mice. Tumors developed in all animals regardless of the cell line injected, but tumor volumes were significantly smaller for both SMAP1-complemented clones compared with HCT116 (P<0.0001, at the time of killing). In vitro, SMAP1 mutations also increased cell clonogenicity (P=0.02-0.04), cell proliferation (P=0.008) by shortening the G2/M phase and decreased cell invasiveness (P=0.03-0.003). In keeping, SMAP1-complemented HCT116 gained several mesenchymal markers (Snail, Slug and vimentin) considered as a hallmark of epithelial-to-mesenchymal transition. These observations are reminiscent of some clinical characteristics of MSI CRCs, notably their larger size and lower rate of metastasis. Our observations suggest that SMAP1 loss-of-function mutations in MSI CRC may contribute to the emerging oncogenic pathway involving abnormal Arf6 regulation.

Interactions between mycoplasmas and the immune system.

Mycoplasmas are a heterogenous group of prokaryotic organisms causing a wide variety of diseases, including autoimmune disorders. Thus, it is not surprising that various mycoplasmas strains, including Mycoplasma arginini, M. arthritidis, M. neurolyticum and M. pulmonis, are able to regulate the immune response. Though some of the studies of the immunomodulatory action of mycoplasmas have been done in vivo, the majority of the investigations have been conducted in vitro. This has led to the recognition that mycoplasmas are polyclonal activators of both B and T cells from several species, acting through MHC-restricted or -unrestricted pathways. Mycoplasma activation not only induces T-cell proliferation but also leads but to the formation of cytotoxic T cells. We, as well as others, have shown that mycoplasma-mediated B-cell activation induces proliferation as well as Ig secretion, and also that mycoplasma stimulation of lymphocytes may result in the production of cytokines. We communicate here our investigations into the effects of an M. arginini strain on the growth and maturation of preactivated B cells. After an initial biological characterization of the M. arginini effects in vitro, we established the protein nature of the growth-supporting activity and proceeded further on to isolate and identify the responsible proteins. The use of lipid- and lipoglycan-free extracts has allowed us to further extend our studies on the biological activities of the proteins from M. arginini and to compare these results with the effects obtained using live organisms. Furthermore, the study was extended to include a characterization of the in vivo-induced effects of live M. arginini. Altogether, the results from these experiments allow us to conclude that M. arginini is a T-cell independent polyclonal B-cell mitogen, mediated by five identified proteins, inducing growth and Ig secretion of both resting and preactivated B cells.

Alternative pathway of complement activation by human lymphoblastoid B and T cell lines.

Other investigators have reported activation of the alternative complement (C) pathway in homologous and heterologous serum by a variety of human lymphoblastoid B cell lines. Their ability to activate the C was associated with Epstein Barr virus transformation of the cells. We report that some human lymphoblastoid T cell lines, lacking EBV-DNA in their genome, do activate the human alternative C pathway with no participation of immunoglobulin or specific antibodies. Nevertheless, activation measured by C3 deposition on the cell surface was weaker than with the B cell lines so far studied; in relation to the RAJl cells, C3 deposition on JURKAT, MOLT 4, HSB2, and 1301 was, respectively, 68, 38, 28, and 19%. Furthermore, C3 deposition on JURKAT T cell line requires D, whereas RAJl cells provide a proteolytic activity able to mimic D, and which, unlike D, can be controlled by serum protease inhibitors. Although the ability to activate the AP seems to be a largely shared property of the human lymphoblastoid B and T cell lines, the situation was strikingly different with normal and mitogen-stimulated lymphocytes or with acute leukemic cells, which led to a negligible AP-dependent C3 deposition compared with the level observed with the lymphoblastoid cell lines. Membrane sialic acid content was determined for every cell tested and revealed no relationship with their ability to activate the AP. The two EBV+ B cell lines tested led to a comparable AP activation regardless of the presence of the C3b receptor.

Biochemical characterization of B cell stimulatory factors for lipopolysaccharide-preactivated B cell blasts: distinction from other known lymphokines.

We have performed a biochemical characterization of B cell stimulatory factors (BSF) produced by Con A-stimulated mouse spleen cells that stimulate growth of lipopolysaccharide (LPS)-preactivated B cells (designated BSF-LPS). Two biochemically distinct forms of BSF-LPS were identified in preparative isoelectric focusing, one acidic form having a pI of 3.9-4.4 and a more basic form with a pI 5.2-5.9. The biochemical heterogeneity of the BSF-LPS activity from Con A-stimulated spleen cells was further demonstrated by ion exchange chromatographies using a fast protein liquid chromatography (FPLC) system. The acidic and the basic forms of BSF-LPS could be totally separated from each other and both are distinct from interleukin-2 (IL-2). Moreover, extending the characterization of the BSF-LPS, together with the use of various murine assay systems for BSF, we could formally exclude that IL-4 or IL-5 accounted for the BSF-LPS activities. In summary, our data provide evidence for the existence of heterogeneous BSF-LPS which maintain growth of LPS-preactivated B cell blasts, and show that these factors can be distinguished from the other lymphokines which have been involved in the control of the cell growth of murine B lymphocytes.

Complement alternative pathway activation by chronic lymphocytic leukemia cells: its role in their hepatosplenic localization.

Using affinity-purified 125I-F(ab')2 anti-human C3, we have investigated the ability of various leukemic cells to activate complement. Lymphocytes from patients with chronic lymphocytic leukemia (CLL) activated the alternative pathway, but cells from patients with other forms of leukemia or normal lymphocytes did not do so. The amount of C3 deposited on the CLL cells was significantly higher in patients with organomegaly (i.e., splenomegaly and/or hepatomegaly). Activation of complement by CLL cells as assessed by C3 deposition on the membrane occurred both in vivo and in vitro and was not related to the N-acetylneuraminic acid content of the membrane.

Functional bioassays for B cell growth factors using polyclonally activated murine spleen B cells.

In this study, we have set up and optimized three distinct T cell-independent polyclonal B cell activation assay systems using highly T cell-depleted murine spleen B cells which were either preactivated in vitro with lipopolysaccharide (LPS) or costimulated with anti-IgM antibodies (anti-mu) or dextran sulfate (DxS). Using these assays, we have investigated the effects of recombinant human or murine interleukins, as well as those of a partially purified T cell-derived factor, designated BSF-LPS. Our results show that none of the interleukins, alone or in combination, is able to maintain growth of the LPS-preactivated B cell blasts, even at the highest concentrations tested, whereas the addition of our BSF-LPS preparation to the cultures significantly increases DNA synthesis. As expected, recombinant murine IL-4 (r mu IL-4) causes a substantial proliferation of anti-mu costimulated B cells. Such anti-mu costimulated B cells also respond, to a lower degree, to recombinant human IL-1 alpha (r hu IL-1 alpha), and do not significantly proliferate upon addition of r mu IL-2, r mu IL-5 or BSF-LPS. On the other hand, r mu IL-5 stimulates very efficiently DxS-costimulated B cells proliferation, whereas r hu IL-1 alpha only exerts a marginal effect; r mu IL-2, r mu IL-4 or BSF-LPS did not maintain growth of DxS-costimulated B cells. We believe that such a thorough investigation of the particular behaviour of activated B cell subpopulations towards various lymphokines provides the background for setting up a valuable bioassay method to differentiate the various interleukins acting on B cells through parallel use of the three distinct T cell-independent polyclonal B cell activation assay systems.

Thymic reticulum in mice. IV. The rosette formation between phagocytic cells of the thymic reticulum and cortical type thymocytes is mediated by complement receptor type three.

A phagocytic cell of the thymic reticulum (P-TR) with dendritic shape recently has been isolated and characterized. We have previously shown that P-TR have an important role to play in the constitution of the thymic microenvironment. Indeed, P-TR are able to produce interleukin 1 and prostaglandin E2, both of which regulate thymocyte activation and proliferation. They are able also to stimulate the proliferation of syngeneic thymocytes enriched in the medullary type. In the present paper, we analyze a close relationship which exists between P-TR and thymocytes of the cortical type. About 25% of P-TR are able to bind to thymocytes and to form rosettes. Rosetting thymocytes represent about 5% of the total population and are PNA+, Lyt 1+2+, H-2-, and sensitive to in vivo steroid treatment. Pretreatment of P-TR with anti-Mac-1, a monoclonal rat IgG antibody against mouse macrophages and specific for complement receptor type three (CR3), abolished rosette formation. Rosette formation also was found to be inhibited by zymosan-treated serum containing the CR3 ligand, C3bi, and by certain sugars, in particular, N-acetyl-D-galactosamine and L-xylose. Our results suggest that rosetting thymocytes bind to CR3 on the P-TR membrane and that sugar constituents of the carbohydrate moieties on the thymocyte surface may serve as a recognition site during the binding process.