Concomitant multipotent and unipotent dental pulp progenitors and their respective contribution to mineralised tissue formation.

Upon in vitro induction or in vivo implantation, the stem cells of the dental pulp display hallmarks of odontoblastic, osteogenic, adipogenic or neuronal cells. However, whether these phenotypes result from genuine multipotent cells or from coexistence of distinct progenitors is still an open question. Furthermore, determining whether a single cell-derived progenitor is capable of undergoing a differentiation cascade leading to tissue repair in situ is important for the development of cell therapy strategies. Three clonal pulp precursor cell lines (A4, C5, H8), established from embryonic ED18 first molars of mouse transgenic for a recombinant plasmid adeno-SV40, were induced to differentiate towards the odonto/osteogenic, chondrogenic or adipogenic programme. Expression of phenotypic markers of each lineage was evaluated by RT-PCR, histochemistry or immunocytochemistry. The clones were implanted into mandibular incisors or calvaria of adult mice. The A4 clone was capable of being recruited towards at least 3 mesodermal lineages in vitro and of contributing to dentin-like or bone formation, in vivo, thus behaving as a multipotent cell. In contrast, the C5 and H8 clones displayed a more restricted potential. Flow cytometric analysis revealed that isolated monopotent and multipotent clones could be distinguished by a differential expression of CD90. Altogether, isolation of these clonal lines allowed demonstrating the coexistence of multipotential and restricted-lineage progenitors in the mouse pulp. These cells may further permit unravelling specificities of the different types of pulp progenitors, hence facilitating the development of cell-based therapies of the dental pulp or other cranio-facial tissues.

Immunohistochemical and molecular analyses of HER2 status in breast cancers are highly concordant and complementary approaches.

BACKGROUND: Immunohistochemistry (IHC) and fluorescent in situ hybridisation (FISH) are currently the most commonly used methods to assess HER2 status. PCR-based assays allow quantitative determination of HER2 amplification (Q-PCR) or overexpression (Q-RT-PCR), but are not routinely used. We evaluated the relevance of Q-RT-PCR for HER2 status determination. METHODS: We compared IHC and Q-RT-PCR in 466 breast tumours. In discordant or equivocal cases, five additional methods (IHC with two other antibodies, FISH, silver in situ hybridisation (SISH) and Q-PCR) were combined to determine HER2 status. Two cases with HER2 intra-tumour heterogeneity were further explored by allelic profiles analysis and HUMARA clonality determination after microdissection. RESULTS: We observed 97.3% concordance between Q-RT-PCR and non-equivocal IHC. Twelve out of 466 cases (3%) revealed discordances between the two methods. The power of Q-RT-PCR to predict HER2 status (defined by seven methods) was similar to that of IHC. Although rare, some discordances between techniques might be due to HER2 intra-tumour heterogeneity and we report two examples, one tumour containing two distinct clones, another tumour consisting of HER2 amplified and non-amplified subclones. CONCLUSION: Q-RT-PCR and IHC are highly concordant methods for HER2 status assessment, and Q-RT-PCR allows a highly reliable quantitative assessment and could be a useful adjunct to IHC.

Changes in allelic imbalances in locally advanced breast cancers after chemotherapy.

In advanced breast cancers, TP53 mutation is highly predictive of complete response to high-dose epirubicin/cyclophosphamide chemotherapy. In these tumours with an altered control of genomic stability, accumulation of chemotherapy-induced genetic alterations may contribute to cell death and account for complete response. To explore the effects of chemotherapy on stability of the tumour genome, allelic profiles were obtained from microdissected tumour samples before and after chemotherapy in 29 unresponsive breast cancers (9 with TP53 mutation). Ninety-four per cent allelic profiles remained unchanged after treatment. Interestingly, 11 profiles (6%) showed important changes after treatment; allelic imbalances significantly increased (four cases) or decreased (seven cases) after chemotherapy in three distinct experiments, two of which using laser microdissected tumour cells. These genetic changes were not linked to the TP53 status, but one tumour showed complete disappearance of TP53-mutated cells in the residual tumour after treatment. Altogether, these observations carry important implications for the clonal evolution of breast cancers treated with DNA-damaging agents, as they point both to the importance of tumour heterogeneity and chemotherapy-driven selection of subclones.

Disruption of T cell regulatory pathways is necessary for immunotherapeutic cure of T cell acute lymphoblastic leukemia in mice.

Acute lymphoblastic leukemia (ALL) is the most common cancer in children. In recent years, the outcome has been globally improved by current therapies, but it remains poor in patients with high, persistent residual disease following the first course of chemotherapy, prompting evaluation of the possible beneficial effects of immunotherapy protocols. In this study, we hypothesized that the disruption of two immunoregulatory pathways controlling the auto-reactive T cell response might synergize with dendritic cell-based immunotherapy of the disease, which is considered to be poorly immunogenic. In this study, we used TAL1xLMO1 leukemia cells adoptively transferred in mice, to generate murine leukemia with poorly immunogenic cells as a model for human T-ALL. Subsequently, these animals were treated with several different immunotherapeutic protocols. We compared the efficiency of a classical, dendritic cell-based immunotherapy (injection of dendritic cells loaded with tumor-derived antigenic products), to a combined treatment associating injection of antigen-loaded dendritic cells and disruption of the two immunoregulatory pathways: CD25+ suppressive T cells and cytotoxic T lymphocyte-associated antigens (CTLA-4). We show that this combined treatment resulted in cure, concomitantly with in vivo generation of immune memory, and TNF-alpha secretion. This study demonstrates that the disruption of these two immunoregulatory pathways synergized with immunostimulation by dendritic cells loaded with tumor-derived antigens, and paves the way for the testing of this combination in clinical trials.

Histochemical and immunohistochemical protocols for routine biopsies embedded in Lowicryl resin.

Tissue processing and analysis require good preservation of both the shape and content of cells. Lowicryl resin is one of the few embedding media that allow good preservation of both tissue architecture and cellular contents. Therefore, different histochemical and immunohistochemical reactions can be applied to semithin sister sections from one biopsy. Further examination of a zone of interest can be carried out under the electron microscope. The hydrophilic property of Lowicryl resins makes possible different histochemical reactions; however, the technique used for paraffin sections must be adapted for each reaction. Antigenic preservation of cells by low temperature embedding allows immunolabeling on either semithin sections or in the zone of interest on ultrathin sections. We have shown the application and adaptation of different histochemical and immunohistochemical reactions on semithin and ultrathin sections from hepatic biopsies that were large, but thin. The variety of techniques that can be used on sister Lowicryl sections of a single biopsy makes this medium useful for extensive pathological studies of precious needle biopsies.

Autonomous rexinoid death signaling is suppressed by converging signaling pathways in immature leukemia cells.

On their own, retinoid X receptor (RXR)-selective ligands (rexinoids) are silent in retinoic acid receptor (RAR)-RXR heterodimers, and no selective rexinoid program has been described as yet in cellular systems. We report here on the rexinoid signaling capacity that triggers apoptosis of immature promyelocytic NB4 cells as a default pathway in the absence of survival factors. Rexinoid-induced apoptosis displays all features of bona fide programmed cell death and is inhibited by RXR, but not RAR antagonists. Several types of survival signals block rexinoid-induced apoptosis. RARalpha agonists switch the cellular response toward differentiation and induce the expression of antiapoptosis factors. Activation of the protein kinase A pathway in the presence of rexinoid agonists induces maturation and blocks immature cell apoptosis. Addition of nonretinoid serum factors also blocks cell death but does not induce cell differentiation. Rexinoid-induced apoptosis is linked to neither the presence nor stability of the promyelocytic leukemia-RARalpha fusion protein and operates also in non-acute promyelocytic leukemia cells. Together our results support a model according to which rexinoids activate in certain leukemia cells a default death pathway onto which several other signaling paradigms converge. This pathway is entirely distinct from that triggered by RAR agonists, which control cell maturation and postmaturation apoptosis.

Alteration of the sphingomyelin/ceramide pathway is associated with resistance of human breast carcinoma MCF7 cells to tumor necrosis factor-alpha-mediated cytotoxicity.

The interference of tumor necrosis factor-alpha (TNF) signaling processes with the acquisition of tumor resistance to TNF was investigated using the TNF-sensitive human breast carcinoma MCF7 cell line and its established TNF-resistant variant (R-A1). The resistance of R-A1 cells to TNF correlated with a low level of p55 TNF receptor expression and an absence of TNF signaling through TNF receptors. Stable transfection of wild-type p55 receptor in R-A1 resulted in enhancement of p55 expression and in partial restoration of TNF signaling, including nuclear factor-kappaB (NF-kappaB) activation. However, the transfected cells remained resistant to TNF-induced apoptosis. Northern blot analysis revealed a comparable induction of manganous superoxide dismutase and A20 mRNA expression in p55-transfected cells and in sensitive MCF7 cells, making it unlikely that these genes are involved in the resistance to TNF-mediated cytotoxicity. While TNF significantly stimulated both neutral and acidic sphingomyelinase (SMase) activities with concomitant sphingomyelin (SM) hydrolysis and ceramide generation in MCF7, it failed to trigger these events in TNF-resistant p55-transfected cells. In addition, the basal SM content was significantly higher in sensitive MCF7 as compared to the resistant counterparts. Furthermore, the TNF-resistant cells tested could be induced to undergo cell death after exposure to exogenous SMase or cell-permeable C6-ceramide. This study also shows that TNF failed to induce arachidonic acid release in p55-transfected resistant cells, suggesting that an alteration of phospholipase A2 activation may be associated with MCF7 cell resistance to TNF. Our findings strongly suggest a role of ceramide in the mechanism of cell resistance to TNF-mediated cell death and may be relevant in elucidating the biochemical nature of intracellular messengers leading to such resistance.

In-vivo effect of the combination of tnf and adriamycin against a human breast cell-line expressing the mdr-phenotype.

The combined effect of tumor necrosis factor alpha (TNF) and adriamycin (ADR) on tumor cell killing was investigated in vivo. The human breast adenocarcinoma ADR-resistant MDA/ADR cells found to be resistant to in vitro TNF lysis and with an apparent index of resistance to ADR of 23 have been used. Treatment of MDA/ADR subcutaneous tumor bearing mice with PBS, TNF, ADR or the combination of TNF/ADR indicate that the combination of TNF and ADR treatment leads to a significant decrease in tumor growth while treatment with TNF was inactive and treatment with ADR was moderately active. Tumor cells were collected from mice treated with TNF/ADR combination and analysed by PCR for MnSOD and TNF ne expression. No induction of the expression of these genes, known to be involved in the regulation of TNF-induced cell killing, was noted following TNF/ADR combination treatment, suggesting that their products were not involved in the observed resistance.

Activation of nuclear protooncogenes and alpha-fetoprotein gene in rat liver during the acute inflammatory reaction.

Nuclear protooncogene and alpha-fetoprotein gene expression is stimulated in hepatocytes during liver regeneration and by various growth factors in vitro. Metabolic adaptation of hepatocytes has been implicated in such gene reprogrammation. We examine here whether induction of an acute inflammation, a physiological situation of important metabolic adjustments, also triggers activation of nuclear oncogenes and of the AFP gene in rat liver. C-fos, c-jun and c-myc mRNA accumulated on Northern blots between 4-12 h of inflammation and the steady-state level of two small alpha-fetoprotein transcripts characteristic of the adult liver increased at 4 h and 24 h of inflammation. In situ hybridization showed accumulation of the mRNA of the four genes studied in all hepatocytes, without any zonal lobular heterogeneity. 3H-histoautoradiography and mitotic counts indicated an inhibition of DNA synthesis and mitosis, prolonged for at least 48 h after inflammation. Thus acute inflammation triggers the activation of nuclear protooncogenes and alpha-feto-protein gene in hepatocytes, but this activation is not followed by passage into the replicative cycle.

Structural and functional studies on the human interleukin-6 receptor. Binding, cross-linking, internalization, and degradation of interleukin-6 by fibroblasts transfected with human interleukin-6-receptor cDNA.

A cDNA coding for the human interleukin-6 receptor (IL-6-R) has been expressed stably in murine NIH/3T3 fibroblasts. Transfected cells exhibited a single class of binding sites for 125I-labeled recombinant human interleukin-6 (125I-rhIL-6) (Kd = 440 pM, 20,000 receptors per cell). Affinity cross-linking of 125I-rhIL-6 to the IL-6-R-expressing NIH/3T3 cells led to the detection of three 125I-rhIL-6-containing protein complexes with molecular masses of 100, 120, and 200 kDa suggesting a complex organization of the IL-6-R in the plasma membrane. IL-6 added to the transfected NIH/3T3 cells exerted growth inhibition. This anti-growth effect was observed by the measurement of cell numbers and ornithine decarboxylase mRNA expression. IL-6-R overexpressing fibroblasts internalized 125I-rhIL-6. Intracellular limited proteolysis of IL-6 could be demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A possible implication of skin fibroblasts in the catabolism of IL-6 is discussed.

Albumin and alpha-fetoprotein gene expression in various nonhepatic rat tissues.

Albumin and alpha-fetoprotein (AFP), two major serum proteins, are synthesized predominantly in the liver and yolk sac of mammals. In the present paper we report on the developmental expression of the corresponding genes in nonhepatic rat tissues. Significant quantities of mature albumin and AFP mRNAs were revealed in kidney, pancreas, heart, and lung of fetal and/or newborn rats using dot blot and Northern blot assays. Very low levels of these mRNA sequences were also detected in adult kidney and pancreas using sensitive RNA-cDNA solution hybridization assays. In situ hybridization analysis revealed that the albumin and AFP gene transcripts are present in the tubular cells of the 20-day-old fetal kidney. In order to elucidate further the mechanisms governing this expression, we studied the chromatin structure and methylation pattern in the 5'-end of these two genes. A faint band, corresponding to a specific DNase I-hypersensitive site upstream from the albumin gene, was detected in the fetal and neonatal kidney nuclei but not in adult kidney. For both genes, a site CG, demethylation of which is correlated with expression in liver and hepatoma cell lines, is highly methylated in fetal kidney even though AFP and albumin genes are expressed. Taken together, these results show the presence of a cell population in the rat kidney that actively transcribes both the albumin and AFP genes. The expression of these genes may be mediated by mechanisms differing in at least some steps from those exerted in the liver.