Thymotaxin, a chemotactic protein, is identical to beta 2-microglobulin.

Thymotaxin, an 11-kilodalton protein chemotactic for rat bone marrow hematopoietic precursors, was purified from media conditioned by a rat thymic epithelial cell line. The NH2-terminal sequence of thymotaxin was identical to that of rat beta 2-microglobulin (beta 2m). Antibodies to beta 2m removed thymotaxin activity from the fraction containing the 11-kilodalton protein. Chemotactic activity was observed with rat plasma beta 2m, human beta 2m, and mouse recombinant beta 2m, further supporting the identity of thymotaxin with beta 2m. The directional migration, as opposed to random movement, of the cells was also confirmed. The only rat bone marrow cells that migrated toward beta 2m were Thy1+ immature lymphoid cells devoid of T cell, B cell, and myeloid cell differentiation markers.

p53 deficiency induces cancer stem cell pool expansion in a mouse model of triple-negative breast tumors.

Triple-negative breast cancer is a heterogeneous disease characterized by the expression of basal cell markers, no estrogen or progesterone receptor expression and a lack of HER2 overexpression. Triple-negative tumors often display activated Wnt/ß-catenin signaling and most have impaired p53 function. We studied the interplay between p53 loss and Wnt/ß-catenin signaling in stem cell function and tumorigenesis, by deleting p53 from the mammary epithelium of K5ΔNßcat mice displaying a constitutive activation of Wnt/ß-catenin signaling in basal cells. K5ΔNßcat transgenic mice present amplification of the basal stem cell pool and develop triple-negative mammary carcinomas. The loss of p53 in K5ΔNßcat mice led to an early expansion of mammary stem/progenitor cells and accelerated the formation of triple-negative tumors. In particular, p53-deficient tumors expressed high levels of integrins and extracellular matrix components and were enriched in cancer stem cells. They also overexpressed the tyrosine kinase receptor Met, a feature characteristic of human triple-negative breast tumors. The inhibition of Met kinase activity impaired tumorsphere formation, demonstrating the requirement of Met signaling for cancer stem cell growth in this model. Human basal-like breast cancers with predicted mutated p53 status had higher levels of MET expression than tumors with wild-type p53. These results connect p53 loss and ß-catenin activation to stem cell regulation and tumorigenesis in triple-negative cancer and highlight the role of Met signaling in maintaining cancer stem cell properties, revealing new cues for targeted therapies.

Analysis of early reconstitution events in the SCID mouse thymus following rat bone marrow cell transplantation.

In this work, we provide comprehensive evidence that sublethally irradiated Thy-1.2 SCID mice can be used as a model system for thymus homing and reconstitution after intravenous transfer of rat bone marrow cells. Full short-term SCID mouse thymus reconstitutions were obtained using a plastic nonadherent low-density rat bone marrow cell subset. Cell counts and flow cytometric analysis showed that at 3 weeks post-transfer the SCID mouse thymus contained up to 41 x 10(6) Thy-1.1high rat lymphoid cells comprising the expected percentages and distribution of CD2+, CD5+, CD3+, alpha beta TCR+ and CD4+ CD8+ cells. As seen on cryostat sections, bone marrow-derived MHC class II+ accessory cells had already developed by 2 weeks post-transfer, preceding the thymocyte expansion apparent at 3 weeks. Thus, analysis of the early events of SCID thymus reconstitution by rat bone marrow cells shows that they closely resemble those described in congenic animals and points out the temporally distinct development of dendritic cells and thymocytes. The SCID mouse-rat chimera model system represents a new in vivo tool for manipulating rat T-cell differentiation from bone marrow-resident precursor cells and in addition supports our previous xenogeneic reconstitution studies performed in organ culture.

Comparative study between biochemical and histological methods and image analysis in liver iron overload.

Iron overload has been measured in 100 hepatic biopsies by three different methods: (i) biochemical assay of the liver iron concentration (LIC), (ii) histological grading (HISTO) and (iii) automated image analysis with a Leitz Texture Analysis System by estimating two parameters, (a) the total iron area (TIA) and (b) the first grey level step (FGLS) at which the iron is detected. Image analysis appears as a specific, sensitive, quick, reproducible and valid method.

Myelin basic protein stimulates the proliferation of astrocytes: possible explanation for multiple sclerosis plaque formation.

In dissociated mouse brain cell cultures we frequently observed an association between myelin basic protein (MBP) positive oligodendrocytes and proliferating astrocytes. When MBP was added in a purified form to the culture medium, it greatly stimulated the proliferation of astrocytes, while other proteins tested did not. This finding allows us to speculate that the gliosis observed in demyelinating diseases or/and in central nervous system (CNS) injury would be due to the mitogenic effect exerted by MBP or its fragments when there is myelin breakdown.

CD34-positive early stages of human T-cell differentiation.

Thymus, the main organ for T lymphopoiesis, requires a permanent influx of progenitors from bone marrow (BM) or fetal liver. An essential question relating to early T-cell development is the identification of the progenitor population which actually homes to the thymus. Recent findings have shown that human multipotent progenitor/stem cells expressing CD34 have the capacity to differentiate into T cells when introduced into a thymic environment. More mature CD34+ bone marrow cells coexpressing CD7 and having a poor myeloid differentiation capacity can also efficiently differentiate into T cells in vitro. These lymphoid committed precursors might be the true thymic repopulating cells. In the thymus, cells with a similar CD34+7+ phenotype include the most primitive thymocyte precursors. CD34+ thymocytes have no myeloid differentiation potential, but may include precursors for natural killer (NK) cells. Interleukin-7 (IL7) is a potent in vitro growth factor for CD34+ thymocytes. Whereas current data do not support a crucial role for IL2, patients with IL2 receptor gamma chain (IL2R gamma) deficiency lack T- and NK cells. The recent demonstration that IL2R gamma is part of the receptor for IL7 strongly suggests that this cytokine plays an essential role in in vivo T lymphocyte and NK development.

Development of mammary gland requires normal beta 1-integrin function.

To study the role of beta 1-integrins in mammary gland development we have generated transgenic mice expressing a dominant negative mutant of the beta 1-integrin chain in the mammary epithelium. The transgenic glands presented a delayed development in pregnancy and lactation due to decreased epithelial cell proliferation and increased apoptosis, whereas at the beginning of lactation, expression of milk proteins, WAP and beta-casein was diminished. In correlation with transgene expression, the basement membrane component, laminin, and the beta 4 integrin were accumulated at the lateral surface of luminal epithelial cells, revealing defects in polarization. Our data show that beta 1-integrins are involved in vivo in the control of proliferation, apoptosis, differentiation, and maintenance of baso-apical polarity of mammary epithelium.

Challenges in the stratification of breast tumors for tailored therapies.

Studying the molecular stratification of breast carcinoma is a real challenge considering the extreme heterogeneity of these tumors. Many patients are now treated following recommendation established at several NIH and St Gallen consensus conferences. However a significant fraction of these breast cancer patients do not need adjuvant chemotherapies while other patients receive inefficacious therapies. High density gene expression arrays have been designed to attempt to establish expression profiles that could be used as prognostic indicators or as predictive markers for response to treatment. This review is intended to discuss the potential value of these new indicators, but also the current weaknesses of these new genomic and bioinformatic approaches. The combined analysis of transcriptomic and genomic alteration data from relatively large numbers of well annotated tumor specimens may offer an opportunity to overcome the current difficulties in validating recently published non overlapping gene lists as prognostic or therapeutic indicators. There is also hope for identifying and deciphering signal transduction pathways driving tumor progression with newly developed algorithms and semi quantitative parameters obtained in simplified in vitro or in vivo models for specific transduction pathways.

Intracellular localization of 12-O-3-N-dansylamino TPA in C3H/10T1/2 mouse cell line.

The fluorescent 12-O-tetradecanoylphorbol-13-acetate (TPA) analogue dansyl-TPA allowed the distribution of fluorescent molecules to be visualized in living mouse fibroblasts. The entry of dansyl-TPA into cells occurred within millisecond time scale and was found largely energy independent. Dansyl-TPA was shown to stain the endoplasmic reticulum, Golgi apparatus, mitochondria and nuclear membrane. In addition, dansyl-TPA fluorescence was found to parallel that obtained with rhodamine-conjugated anti-alpha tubulin. The intracellular location of the dansyl-TPA fluorescence can be taken into account to explain the pleiotropic action of TPA in the cells.

Immunocytochemical quantitative study of alpha-fetoprotein in normal and neoplastic neural development.

The presence of alpha-fetoprotein (AFP) was studied in developing nervous tissues: rat cerebral cortex and neuroepithelial derivatives of a murine teratocarcinoma. AFP was localized by an indirect immunohistoperoxidase technique. Using serial dilutions of anti-AFP antibodies, the end-points of staining (minimal concentration of antibodies giving a positive labelling) were established. These points enabled the evaluation of the amount of stained AFP at different periods of development. In rat cerebral cortex, AFP showed one maximum amount on the 19th-20th day 'postcoitum' (in moderately differentiated structures), and two minima on the 13th-14th day (in primitively differentiated ones) and the 28th-29th day (in well-differentiated ones), respectively. Similarly, one maximum and two minima were established in the derivatives of teratocarcinoma imitating normal neural development.

Tumor progression: the role of cadherins and integrins.

Established cadherin and integrin-containing adherens junctions maintain the integrity of normal tissues. Signalling via adhesion-molecule systems is an important factor in the control of cellular growth and differentiation. In transformed cells, destructive changes in the adhesion systems lead to abnormal relationships among neighbouring cells and the extracellular matrix. Adhesion molecules may prevent tumour progression by firmly attaching cells to each other, and by anchoring them in the extracellular matrix. In addition, cadherins and integrins may have a direct role in tumour suppression by participating in growth control. Dissemination of cancer cells, i.e. invasion and metastasis, requires movement of cells, as well as adhesion to extracellular matrices and to other cells. Particular integrins have been implicated in several aspects of this multistep process. In this article, the data on the possible roles of cadherins and integrins in tumor progression are summarized.

Aspects of haemopoietic cell dynamics: ontogeny and targeted migration.

In the developing avian and mammalian embryo, haemopoietic cells appear first in transient foci whose function is restricted to discrete periods of embryogenesis. These foci are essentially represented by the yolk sac, intraembryonic dispersed foci and the liver. Haemopoietic cells then repopulate the developing spleen, thymus and bone marrow, organs which persist and develop after birth. In the present review, we describe a number of possible mechanisms controlling specific adhesion, oriented migration and invasiveness of haemopoietic cells. One concerns the high specificity of the interactions of homing receptors on the surface of haemopoietic cells with determinants on vascular endothelium and/or thymic epithelium. A second is the importance of the presence of some macromolecules in the extracellular matrix, such as fibronectin, collagen, laminin and elastin. These components can interact with the haemopoietic cells (and/or induce chemotaxis) via the existence of specific receptors on the surface of the haemopoietic cells. Another mechanism is the activation of the haemopoietic cells through the interactions of cell-chemotactic factor, cell-extracellular matrix and/or cell-thymic epithelium. This activation can lead to: 1) the expression of new specific cell-surface receptors for the target foci; 2) the secretion of specific protease and glycosidase systems active upon the extracellular matrix; and 3) the differentiation of these cells in the thymus.

Histochemistry of oxidative metabolism in epididymal epithelium of mouse.

The activities of cytoplasmic and mitochondrial dehydrogenases were assessed histochemically in the lining epithelia of five segments of the proximal part and the medial and distal parts of the mouse epididymis. The cytochemical evaluation of enzyme activities revealed by nitro BT or tetranitro BT was well supported by the relative transmittance values. In the principal cells of the proximal part, the activities of dehydrogenases differed according to histological segmentation. In the medial and distal parts of the duct, a progressive increase in the intensity of all the reactions was observed. The "goblet cells" with apical nuclei in the proximal part and the "clear cells" in the medial and distal parts showed higher activities than the adjacent principal cells.

Overproduction and secretion of beta 2-microglobulin by a rat thymic epithelial cell line that expresses MHC class I heavy chain.

Major histocompatibility complex (MHC) class I antigens are constituted of dimers consisting of a peripheral light chain, beta 2-microglobulin (beta 2m) and a transmembrane heavy chain whose cell surface expression depends on its assembly with beta 2m. In contrast, soluble beta 2m can be secreted in the absence of heavy chain expression. The presence of beta 2m in medium conditioned by a rat thymic epithelial cell line, IT45-R1 (IT45) prompted us to investigate whether beta 2m could be secreted by cells that express MHC class I antigens. IT45 cells produce three to five times more beta 2m in the culture supernatant than another rat thymic epithelial cell line, IT26-R21 (IT26). The IT45 cell line exported beta 2m through a constitutive pathway of secretion, as indicated by the kinetics of production and localization of intracellular beta 2m. Although cells from the IT45 cell line expressed a much higher amount of beta 2m as compared to IT26 and NBT II cells (a rat bladder epithelial cell line), all three of these cell lines expressed the same amount of membrane and intracellular MHC class I heavy chain. These data are thus consistent with a constitutive secretion of beta 2m dependent upon an overexpression of MHC class I light chain as compared to the heavy chain. The amount of beta 2m mRNA and the ratio of beta 2m versus MHC class I heavy chain transcripts were higher in IT45 than in IT26 cells, indicating that overexpression of beta 2m in IT45 cells could be due to an enhanced level of beta 2m mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Surface distribution of wheat germ agglutinin binding sites of human melanoma cell lines with low and high tumorigenicity.

Wheat germ agglutinin (WGA) binding patterns of human malignant melanoma cell lines with a high or a low ability to grow subcutaneously in nude mice were compared. SDS-PAGE analysis of WGA binding glycoproteins revealed similar qualitative but different quantitative profiles. Striking differences were observed in the density of WGA binding sites, twice as high in the low tumorigenic cell line as in the high tumorigenic cell line. Differences were also observed in the topographical organization of these WGA binding sites patched on the low tumorigenic cell line and diffuse on the high tumorigenic cell line. Fluorescence photobleaching measurements showed clear-cut differences in their motion patterns.

Growth defects induced by perturbation of beta1-integrin function in the mammary gland epithelium result from a lack of MAPK activation via the Shc and Akt pathways.

Adhesion to extracellular matrix (ECM) induces intracellular signals that modulate cell proliferation, survival and differentiation. To study signalling events triggered by cell-ECM interactions in vivo we used transgenic mice exhibiting reduced mammary epithelial cell proliferation and increased apoptosis rates during the growth phase in pregnancy and lactation due to expression of a beta1-integrin dominant-negative mutant in the mammary gland epithelium. Here we show that ERK and JNK MAPKs were markedly less activated in lactating transgenic glands thereby accounting for the growth defects. The FAK pathway was not affected suggesting a mechanism of activation additional to the ECM signal. On the contrary, the significant decrease of Shc phosphorylation, Grb2 recruitment and the reduced phosphorylation level of Akt Thr308 and Akt substrates FKHR and Bad detected in transgenic glands show that activation of the Shc and the Akt pathways require intact cell-ECM interactions. These results provide an insight into the mechanisms of growth control by integrin-mediated adhesion that operate in vivo.

Myoepithelial cell differentiation in the developing mammary gland: progressive acquisition of smooth muscle phenotype.

The most important portion of the mammary gland development occurs postnatally, with distinct periods of intensive morphogenesis taking place between birth and puberty and during pregnancy and lactation. To characterize the differentiation process of mammary myoepithelial cells, we have studied the expression patterns of several smooth muscle phenotypic markers, including contractile proteins, alpha-smooth muscle-actin (alpha-SM-actin), smooth muscle myosin heavy chains (SM-MHC), and calponin; components of cell-extracellular matrix adherens junctions, phosphoglucomutase-related protein (PGM), vinculin variants, integrin subunits; and laminin variant chains in the developing rat mammary gland using immunofluorescence microscopy. alpha-SM-actin- and SM-MHC-positive cells were found first in newborn animals, while calponin, PGM and alpha1 integrin subunit began to be expressed in prepubertal animals (1.5 weeks). Vinculin, beta1 and alpha3 integrin subunits were largely confined to the basal cell layer at all developmental stages examined with greater staining starting at 1.5 weeks. Meta-vinculin was identified only in myoepithelial cells of the lactating gland. gamma1 laminin chain was present in the mammary gland basement membrane throughout development, while the beta2 chain was revealed in 3-week-old animals and accumulated later in postpubertal animals (7 weeks). Similarly, beta2 laminin chain was absent from the forming alveoli basement membrane in pregnant rats and started to accumulate in the lactating gland. In addition to temporal changes, we have observed spatial differences in the distribution of the phenotypic markers. Both in pre- and in postpubertal animals, alpha-SM-actin- and SM-MHC-positive cells of the growing ductal ends contained low amounts if any of calponin, PGM, and beta2 laminin chain. We conclude that during postnatal development, mammary myoepithelial cells progressively acquire a differentiated phenotype as revealed by the expression of various smooth muscle markers. Maturation of the myoepithelial cells is accompanied by upregulation of the smooth muscle integrin expression followed by accumulation of beta2-containing laminin variant. Thus, changes in adhesion system parallel with the myoepithelial cell differentiation.

Perturbation of beta1-integrin function alters the development of murine mammary gland.

The expression of a transgene coding for a chimeric molecule, containing the cytoplasmic and transmembrane domains of the beta1-integrin chain and the extracellular domain of the T-cell differentiation antigen CD4, was targeted to the mouse mammary gland by the mouse mammary tumor virus (MMTV) promoter. The chimera does not interact with the extracellular ligands; however, its expression in cultured cells was shown to interfere with focal adhesion kinase (FAK) phosphorylation following ligation of endogenous beta1-integrin. Therefore, expression of the transgenic protein on the cell surface should uncouple adhesion from intracellular events associated with the beta1-cytoplasmic domain and thus perturb beta1-integrin functions. Although most of the transgenic females were able to lactate, their mammary glands had a phenotype clearly distinct from that of wild-type mice. At mid-pregnancy and the beginning of lactation, transgenic glands were underdeveloped and the epithelial cell proliferation rates were decreased, while the apoptosis levels were higher than in wild-type glands. In lactation, the amounts of the whey acidic protein (WAP) and beta-casein gene transcripts were diminished, and the basement membrane component, laminin and the beta4-integrin chain accumulated at the lateral surface of luminal epithelial cells, revealing defects in polarization. Our observations prove that in vivo, beta1-integrins are involved in control of proliferation, apoptosis, differentiation and maintenance of baso-apical polarity of mammary epithelial cells, and therefore are essential for normal mammary gland development and function.