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Biomedical subjects

E Reichmann

Publications and source records attributed to E Reichmann.

At least 19 recordsLinked to original sources
PubMed2006

Impact of carbon dioxide versus air pneumoperitoneum on peritoneal cell migration and cell fate.

BACKGROUND: Postoperative systemic immune function is suppressed after open abdominal surgery, as compared with that after minimally invasive abdominal surgery. As a first line of defense, peritoneal macrophages (PMo) and polymorphonuclear neutrophil granulocytes (PMNs) are of primary importance in protecting the body from microorganisms. Previous studies have shown changes in these cell populations over time after open versus laparoscopic surgery. This study aimed to investigate the dynamics of cell recruitment and clearance of peritoneal cells. METHODS: Female NMRI mice (33 +/- 2 g) were randomly assigned to carbon dioxide (CO2) or air insufflation. Intravasal cells with phagocytic capabilities were selectively stained by intravenous injection of the fluorescent dye PKH26 24 h before surgery. Gas was insufflated into the peritoneal cavity through a catheter, and the pneumoperitoneum was maintained for 30 min. Peritoneal lavage was performed 1, 3, 8, or 24 h after surgery. Apoptotic cells were assessed by flow cytometry using a general caspase substrate. RESULTS: The total peritoneal cell count did not differ between groups. The PKH26-positive PMo level was significantly increased after CO2, as compared with air, at 1 h and 24 h. The ratio of apoptotic PMo did not differ between the groups. In the peritoneal lavage, polymorphonuclear leukocytes (PMNs) were tripled in the air group, as compared with the CO2 group, whereas the ratio of apoptotic PMNs was significantly decreased. There was a higher fraction of PKH26-positive PMNs after air exposure, as compared with that after CO2. CONCLUSIONS: Air exposure triggered a higher transmigration rate of PMNs from the blood compartment into the peritoneal cavity and decreased PMN apoptosis, as compared with CO2. The lower proportion of PKH26-positive peritoneal macrophages in the air group might have been attributable to a higher inflammatory stimulation than in the CO2 group, leading to increased emigration of PMo to draining lymph nodes. All the findings underscore a complex cell-specific regulation of cell recruitment and clearance in the peritoneal compartment.

Air↗
PubMed2006

Functional characterization of cell hybrids generated by induced fusion of primary porcine mesenchymal stem cells with an immortal murine cell line.

Bone marrow mesenchymal stem cells (MSC) integrate into various organs and contribute to the regeneration of diverse tissues. However, the mechanistic basis of the plasticity of MSC is not fully understood. The change of cell fate has been suggested to occur through cell fusion. We have generated hybrid cell lines by polyethylene-glycol-mediated cell fusion of primary porcine MSC with the immortal murine fibroblast cell line F7, a derivative of the GM05267 cell line. The hybrid cell lines display fibroblastic morphology and proliferate like immortal cells. They contain tetraploid to hexaploid porcine chromosomes accompanied by hypo-diploid murine chromosomes. Interestingly, many hybrid cell lines also express high levels of tissue-nonspecific alkaline phosphatase, which is considered to be a marker of undifferentiated embryonic stem cells. All tested hybrid cell lines retain osteogenic differentiation, a few of them also retain adipogenic potential, but none retain chondrogenic differentiation. Conditioned media from hybrid cells enhance the proliferation of both early-passage and late-passage porcine MSC, indicating that the hybrid cells secrete diffusible growth stimulatory factors. Murine F7 cells thus have the unique property of generating immortal cell hybrids containing unusually high numbers of chromosomes derived from normal cells. These hybrid cells can be employed in various studies to improve our understanding of regenerative biology. This is the first report, to our knowledge, describing the generation of experimentally induced cell hybrids by using normal primary MSC.

Adipogenesis↗
PubMed2005

Early peritoneal macrophage function after laparoscopic surgery compared with laparotomy in a mouse mode.

BACKGROUND: The authors previously demonstrated postoperative preservation of the immune function measured by delayed-type skin reaction and tumor growth after laparoscopic surgery, as compared with laparotomy. For further elucidation of the origin of the demonstrated immune preservation, peritoneal macrophage (PMo) function was investigated 1 h after different surgical procedures. METHODS: Female NMRI mice were divided into five groups: anesthesia only, abdominal skin incision, laparotomy, peritoneal carbon dioxide (CO2) insufflation, and peritoneal air insufflation. Escherichia Coli phagocytosis, tumor necrosis factor-alpha (TNF-alpha), transforming growth factor-beta1 (TGF-beta1), and interleukin-10 (IL-10) release of isolated PMo were investigated. RESULTS: All invasive interventions reduced the PMo phagocytosis by factors of approximately 2 to 4.7, as compared with the sham control group. Spontaneous ex vivo TNF-alpha release was significantly increased whenever the abdominal cavity was exposed to ambient air. The macrophage's ability to release TNF-alpha after E. coli exposure was diminished in the abdominal air exposure groups, as compared with the CO2 insufflation group. CONCLUSION: Reduced phagocytosis 1 h after surgical interventions suggests a contribution of PMo to the altered immune function. When exposed to CO2, PMo show a decreased basal TNF-alpha release. However, PMo also show an increased TNF-alpha release after a second immune stimulation (E. coli), suggesting a greater competency of interaction in an immune defense reaction after CO2 exposure.

Animals↗
PubMed2000

Fas-dependent tissue turnover is implicated in tumor cell clearance.

The apoptosis-inducing Fas receptor has been shown to be down-regulated in various types of tumors, while its ligand (FasL) appears to be frequently up-regulated. Here we provide evidence that there is a strong selective pressure in vivo against Fas-expressing, tumorigenic NIH3T3 cells, favoring survival, proliferation and eventually tumor formation by Fas-negative cells. Importantly, re-expression of Fas in these cells results in either the complete abolishment of tumor development, or in a significant extenuation of the latency period of tumor outgrowth. In addition, we found that environmental conditions which prevail during tumorigenesis, such as limiting amounts of survival factors and the lack of cell adhesion, are markedly sensitizing tumor cells to Fas-mediated suicide. Our data suggest that in addition to T cell-mediated immune responses, mechanisms of Fas-dependent tissue turnover are also centrally implicated in tumor cell clearance.

3T3 Cells↗
PubMed1999

Phosphorylation and free pool of beta-catenin are regulated by tyrosine kinases and tyrosine phosphatases during epithelial cell migration.

Cell migration requires precise control, which is altered or lost when tumor cells become invasive and metastatic. Although the integrity of cell-cell contacts, such as adherens junctions, is essential for the maintenance of functional epithelia, they need to be rapidly disassembled during migration. The transmembrane cell adhesion protein E-cadherin and the cytoplasmic catenins are molecular elements of these structures. Here we demonstrate that epithelial cell migration is accompanied by tyrosine phosphorylation of beta-catenin and an increase of its free cytoplasmic pool. We show further that the protein-tyrosine phosphatase LAR (leukocyte common antigen related) colocalizes with the cadherin-catenin complex in epithelial cells and associates with beta-catenin and plakoglobin. Interestingly, ectopic expression of protein-tyrosine phosphatase (PTP) LAR inhibits epithelial cell migration by preventing phosphorylation and the increase in the free pool of beta-catenin; moreover, it inhibits tumor formation in nude mice. These data support a function for PTP LAR in the regulation of epithelial cell-cell contacts at adherens junctions as well as in the control of beta-catenin signaling functions. Thus PTP-LAR appears to play an important role in the maintenance of epithelial integrity, and a loss of its regulatory function may contribute to malignant progression and metastasis.

Animals↗
PubMed1999

Oncogenic Ras inhibits Fas ligand-mediated apoptosis by downregulating the expression of Fas.

Tumor growth is the result of deregulated tissue homeostasis which is maintained through the delicate balance of cell growth and apoptosis. One of the most efficient inducers of apoptosis is the death receptor Fas. We report here that oncogenic Ras (H-Ras) downregulates Fas expression and renders cells of fibroblastic and epitheloid origin resistant to Fas ligand-induced apoptosis. In Ras-transformed cells, Fas mRNA is absent. Inhibition of DNA methylation restores Fas expression. H-Ras signals via the PI 3-kinase pathway to downregulate Fas, suggesting that the known anti-apoptotic effect of the downstream PKB/Akt kinase may be mediated, at least in part, by the repression of Fas expression. Thus, the oncogenic potential of H-ras may reside on its capacity not only to promote cellular proliferation, but also to simultaneously inhibit Fas-triggered apoptosis.

3T3 Cells↗
PubMed1996

TGF-beta1 and Ha-Ras collaborate in modulating the phenotypic plasticity and invasiveness of epithelial tumor cells.

Metastasis of epithelial tumor cells can be associated with the acquisition of fibroblastoid features and the ability to invade stroma and blood vessels. Using matched in vivo and in vitro culture systems employing fully polarized, mammary epithelial cells, we report here that TGF-beta1 brings about these changes in Ras-transformed cells but not in normal cells. When grown in collagen gels in the absence of TGF-beta, both normal and Ras-transformed mammary epithelial cells form organ-like structures in which the cells maintain their epithelial characteristics. Under these conditions, treatment of normal cells with TGF-beta results in growth arrest. The same treatment renders Ras-transformed epithelial cells fibroblastoid, invasive, and resistant to growth inhibition by TGF-beta. After this epithelial-fibroblastoid conversion, the Ras-transformed cells start to secrete TGF-beta themselves, leading to autocrine maintenance of the invasive phenotype and recruitment of additional cells to become fibroblastoid and invasive. More important, this cooperation of activated Ha-Ras with TGF-beta1 is operative during in vivo tumorigenesis and, as in wound healing processes, is dependent on epithelial-stromal interactions.

Animals↗
PubMed1996

The estrogen-dependent c-JunER protein causes a reversible loss of mammary epithelial cell polarity involving a destabilization of adherens junctions.

Members of the epidermal growth factor (EGF) receptor family are known to be specifically involved in mammary carcinogenesis. As a nuclear target of activated receptors, we examined c-Jun in mammary epithelial cells. For this, we used a c-JunER fusion protein which was tightly controlled by estrogen. Activation of the JunER by hormone resulted in the transcriptional regulation of a variety of AP-1 target genes. Hormone-activated JunER induced the loss of epithelial polarity, a disruption of intercellular junctions and normal barrier function and the formation of irregular multilayers. These changes were completely reversible upon hormone withdrawal. Loss of epithelial polarity involved redistribution of both apical and basolateral proteins to the entire plasma membrane. The redistribution of E-cadherin and beta-catenin was accompanied by a destabilization of complexes formed between these two proteins, leading to an enrichment of beta-catenin in the detergent-soluble fraction. Uninduced cells were able to form three-dimensional tubular structures in collagen I gels which were disrupted upon JunER activation, leading to irregular cell aggregates. The JunER-induced disruption of tubular structures was dependent on active signaling by growth factors. Moreover, the effects of JunER could be mimicked in normal cells by the addition of acidic fibroblast growth factor (aFGF). These data suggest that a possible function of c-Jun in epithelial cells is to modulate epithelial polarity and regulate tissue organization, processes which may be equally important for both normal breast development and as initiating steps in carcinogenesis.

Animals↗
PubMed1995

Progression of carcinoma cells is associated with alterations in chromatin structure and factor binding at the E-cadherin promoter in vivo.

E-cadherin has been identified as a tumor (invasion) suppressor gene, which is mutated in 50% of diffuse-type human gastric carcinomas. In other carcinomas, the expression of E-cadherin is down-regulated in the poorly differentiated cells such as from breast, bladder, lung and colon. We have here examined the in vivo properties of the genomic E-cadherin promoter in well and poorly differentiated carcinoma cell lines in order to gain insights into the mechanisms of E-cadherin down-regulation in tumors. In vivo footprinting analysis revealed that positive regulatory elements of the E-cadherin promoter (a GC-rich region, the CCAAT-box and a palindromic element) are specifically bound by transcription factors in E-cadherin-expressing but not in non-expressing cells. The tested cell systems include more than a dozen carcinomas cell lines as well as mammary epithelial cells where E-cadherin expression can be switched off by activation of a Fos-estrogen receptor fusion protein and rhabdomyosarcoma cells where E-cadherin expression was induced by transfection with E1A. Mapping of DNase I hypersensitive sites showed that the chromatin structure in the promoter region is loosened in expressing but condensed in non-expressing cells. Furthermore, the endogenous E-cadherin promoter is specifically methylated at CpG sites in the undifferentiated cells. We also show that the in vivo properties of the promoter in E-caherin-negative carcinoma cells are similar as in mesenchymal cells, i.e. fibroblasts or sarcoma cells. These data suggest that silencing of the E-cadherin promoter during epithelialmesenchymal transition and tumor progression is due to a loss of factor binding in vivo and to chromatin rearrangement in the regulatory region.

Animals↗
PubMed1994

The effects of the neuN and neuT genes on differentiation and transformation of mammary epithelial cells.

Overexpression of the proto-oncogene product, p185neuN, in a non-tumorigenic mammary epithelial line (31E) facilitates aspects of lactogenic differentiation. Formation of branching cords and induction of beta-casein synthesis by 31E cells normally require co-culture of these cells with fibroblasts, or the presence of collagen or fibronectin. In contrast, 31E cells expressing p185neuN spontaneously form branching cords when grown on tissue culture plastic and can synthesize beta-casein in the absence of exogenous substrates or feeder layers. Under these conditions, the cells deposit laminin and fibronectin, indicating a possible role for p185neuN in the deposition of extracellular matrix proteins. Overexpression of the corresponding oncogene product, p185neuT, has markedly different effects. Expression of p185neuT does not facilitate the formation of branching cords or the synthesis of beta-casein when grown on tissue culture plastic, although these cells do deposit laminin and fibronectin. Confocal microscopy indicates a significant difference in the distribution of laminin and fibronectin in 31E cells expressing p185neuT compared to those expressing p185neuN. The effects of p185neuN and p185neuT expression on cell transformation depend on cell type. Expression of both p185neuN and p185neuT increases anchorage-independent growth of 31E cells, but only p185neuT induces anchorage-independent growth of NIH 3T3 fibroblasts. This lineage specificity in the action of p185neuN may be related to observations that overexpression of p185c-erbB-2 (the human homologue of p185neuN) is only associated with the development of human epithelial cancers. The effects of p185neuN on laminin deposition by 31E cells may be relevant to the transforming ability of p185neuN, since laminin can induce anchorage-independent growth of mouse mammary cells. These results suggest that p185neuN and p185neuT could exert their effects on differentiation and transformation of mammary epithelial cells in part by promoting the deposition of extracellular matrix proteins.

Animals↗
PubMed1994

Oncogenes and epithelial cell transformation.

More than 80% of the tumors occurring in man are carcinomas. Despite the prevalence of these epithelial tumors most in vitro studies of oncogenesis have employed mesenchymal rather than epithelial cells. As a result, a detailed understanding of the molecular mechanisms of carcinoma formation and an exact definition of the malignant transformed epithelial phenotype are lacking. Taking into account that the genesis of a carcinoma involves alterations in multiple genes, giving rise to complex phenotypic changes, the question arises whether each single, mutational step can be correlated with distinct alterations of the epithelial phenotype. To approach this question, oncogenes that are both critically positioned in growth factor receptor-driven signaling pathways and relevant in carcinoma development, such as ras, src, myc and fos, have been expressed in epithelial cells. The results indicate that epithelial cells transformed by oncogenes in vitro alter their gene expression programs, thereby losing certain features of cell polarity and cell adhesion. However, as with true tumor cells, these changes occur in distinct combinations, to different degrees and are influenced by the local environment of the cells.

Cell Transformation, Neoplastic↗
PubMed1993

T1, an immunoglobulin superfamily member, is expressed in H-ras-dependent epithelial tumours of mammary cells.

T1 is a glycosylated protein in the carcinoembryonic antigen (CEA) family of tumour marker molecules. It was originally identified by virtue of its transient induction after the expression of p21H-ras in NIH3T3 fibroblasts. Here we show that the T1 gene is activated in mammary adenocarcinomas of transgenic mice harbouring an H-ras transgene under the control of the mammary-specific whey acidic protein (WAP) promoter. By contrast, T1 mRNA was not, or only faintly, detectable in mammary carcinomas of transgenic mice bearing a WAP-myc transgene. Thus, T1 overexpression does not appear to be a general tumour-specific phenomenon. A dependence of T1 gene expression on the action of p21H-ras is suggested by the observation of T1 mRNA in nude mouse tumours generated from H-ras-transformed cultured mammary epithelial cells. Interestingly, activation of the T1 gene is also found during the maturation of the mammary gland (3-4 weeks after birth), whereas it is absent during its terminal differentiation in pregnancy and lactation. This expression pattern suggests a role for the secreted T1 glycoprotein in the phase of epithelial proliferation of the mammary gland. It appears that p21H-ras-induced transformation of mammary epithelial cells mimics the situation occurring in puberty. In both developmental stages the T1 glycoprotein might affect cell interactions of the proliferating epithelial cells with the surrounding stroma. It might thus promote ductal outgrowth in gland maturation as well as invasive growth of p21H-ras-transformed mammary epithelial cells.

Adenocarcinoma↗
PubMed1992

Activation of an inducible c-FosER fusion protein causes loss of epithelial polarity and triggers epithelial-fibroblastoid cell conversion.

As a novel approach to studying the modulation of the polarized epithelial phenotype, we have expressed c-Fos and c-Myc estrogen receptor fusion proteins (c-FosER and c-MycER) in mammary epithelial cells. The hybrid proteins could be activated by estrogen for defined time periods and after the cells had achieved their fully polarized organization. Activation of c-MycER deregulated proliferation but did not affect epithelial polarity. Short-term activation of c-FosER induced the reversible loss of morphological and functional cell polarity. In contrast, long-term stimulation of c-FosER caused the cells to depolarize irreversibly, to invade collagen gels, and to undergo epithelial-fibroblastoid cell conversion. Our data suggest that Fos proteins are important in modulating the epithelial phenotype both in normal tissue development and in invasive processes.

Animals↗
PubMed1991

Mammary epithelial differentiation in vitro: minimum requirements for a functional response to hormonal stimulation.

Mammary epithelial differentiation is the culmination of responses to a complex sequence of hormonal stimuli. An in vitro model for this process should retain the basic features of in vivo epithelial differentiation. The IM-2 mouse mammary cell line responds to lactogenic hormone stimulation by synthesizing the milk protein beta-casein. Epithelial and fibroblastic clones derived from IM-2 lack this ability, but cocultures of these clones regain responsiveness to lactogenic hormone stimulation. Studies of the epithelial cell clone 31E under various culture conditions reveal that the role of fibroblastic cells in supporting synthesis and secretion of beta-casein can be supplanted by culture in filter chambers without addition of exogenous extracellular matrix components. Electron microscopic and immunofluorescence studies show that, under these conditions, 31E epithelial cells exhibit the morphology and intercellular organization characteristic of mammary epithelium. Transepithelial electrical resistance measurements indicate that the cells are well polarized. Analysis of glucose metabolism is consistent with this polarization; glucose is utilized from the basal chamber, and lactate is excreted into the basal chamber. Immunoblot analysis demonstrates the vectorial protein secretion expected of polarized mammary epithelium: laminin is secreted into the basal chamber, whereas beta-casein is secreted into the apical chamber in response to lactogenic hormone stimulation from the lower chamber. Thus, the maintenance of a polarized intercellular organization that permits access of the basolateral cell surface to nutrients is sufficient for a pure culture of an established mammary epithelial cell clone to retain differentiated epithelial function in vitro.

Animals↗
PubMed1989

New mammary epithelial and fibroblastic cell clones in coculture form structures competent to differentiate functionally.

We have established and characterized a spontaneously immortalized, nontumorigenic mouse mammary cell line, designated IM-2. IM-2 cells synthesize large amounts of the milk protein beta-casein upon addition of lactogenic hormones. The induction of beta-casein occurs rapidly and does not require any exogenous extracellular matrix components. The IM-2 cell line is morphologically heterogeneous and could be separated into cell clones with epithelial and fibroblastic characteristics. In monoculture, none of the epithelial clones could be induced to synthesize caseins. Coculture of epithelial and fibroblastic clones, however, rendered the epithelial cells competent to differentiate functionally; the addition of lactogenic hormones to these cocultures resulted in the synthesis of beta-casein in amounts comparable to that seen with the original IM-2 line. Using this unique cell system, we have investigated the interrelationships between different steps in differentiation leading to hormone-induced casein production. Independent of hormones, epithelial-fibroblastic cell contacts led to the formation of characteristic structures showing the deposition of laminin. We found that the epithelial cells located in these structures also exhibited significantly increased levels of cytokeratin intermediate filament polypeptides. Double immunofluorescence revealed that the cells inducible by hormones to synthesize casein, colocalized exactly with the areas of laminin deposition and with the cells showing greatly intensified cytokeratin expression. These results suggest that hormone-independent differentiation events take place in response to intercellular epithelial-mesenchymal contacts. These events in turn bring about a state of competence for functional differentiation after lactogenic hormonal stimulation.

Animals↗
PubMed1988

v-myc alters the response of a cloned mouse mammary epithelial cell line to lactogenic hormones.

Several oncogenes have now been implicated in mammary carcinogenesis. We investigated the phenotypic effects of expressing three representative oncogenes in mammary epithelial cells. v-myc (coding for a nuclear protein), v-Ha-ras (a G-protein homologue) and v-fgr (a tyrosine kinase) genes were introduced into the nontumorigenic clone 14 of the mouse mammary epithelial cell line COMMA-1D. Their effects upon growth and differentiation were determined. Anchorage-independent growth was induced by all three oncogenes with low efficiency. v-Ha-ras and v-fgr induced tumorigenicity in nude mice. The effect of oncogenes upon parameters unique to mammary epithelial cells in vitro was assayed. Both v-myc and v-fgr abolished the ability of clone 14 to grow as three-dimensional branching structures in hydrated collagen gel. v-fgr completely and v-myc partially inhibited the expression of the epithelium specific cytokeratins. Clone 14 can be induced to produce the beta-casein milk protein by the combination of the lactogenic hormones, dexamethasone, insulin, and PRL. Introduction of v-myc into clone 14 cells resulted in an estimated 50-fold increased induction of beta-casein protein and at least a 60-fold increase in beta-casein mRNA. The number of cells stained with anti-beta casein antibodies also showed a 10-fold increase after v-myc introduction. This still required the synergistic action of all three lactogenic hormones. Thus v-myc can alter the normal response of mammary epithelial cells to lactogenic hormones.

Animals↗