Isolation of mouse mammary epithelial progenitor cells with basal characteristics from the Comma-Dbeta cell line

A mouse mammary epithelial cell line with morphogenetic properties in vivo, Comma-Dbeta, was used to isolate and to characterize mammary progenitor cells. We found that a homogeneous cell population expressing high surface levels of stem cell antigen 1 (Sca-1) was able to give rise in vivo to ductal and alveolar structures comprising luminal secretory and basal myoepithelial cells. Unlike the Sca-1(high), the Sca-1(neg/low) cell population displayed a reduced morphogenetic potential. The Sca-1(high) cells presented moderate CD24, high CD44 and alpha6 integrin surface levels, expressed basal cell markers p63, keratins 5 and 14, but no luminal and myoepithelial lineage markers. In culture, the Sca-1(high) cells generated identical daughter cells that retained their in vivo developmental potential, indicating that these cells were maintained by self-renewal. Plated at clonogenic density in Matrigel, Sca-1(high) cells formed spheroids that included luminal and myoepithelial cells. Thus, the isolated Sca-1(high) basal cells possess several features of stem/progenitor cells, including specific markers, self-renewal capacity, and the ability to generate the two major mammary lineages, luminal and myoepithelial. These data provide evidence for the existence of basal-type mouse mammary progenitors able to participate in the morphogenetic processes characteristic of mammary gland development.     

Epidermal and hepatocyte growth factors stimulate chemotaxis in an intestinal epithelial cell line

The migration of intestinalcells is important in the development and maintenance of normalepithelium, in a process that may be regulated by growth factors andcytokines. Although a number of growth factor receptors are expressedby intestinal cells, little progress has been made toward assignment offunctional roles for these ligand-receptor systems. This study comparesseveral growth factors and cytokines for their chemoattraction of the mouse small intestinal epithelial cell line. Epidermal and hepatocyte growth factors stimulated a rapid 30-fold chemotaxis of cells withdelayed threefold migration toward transforming growth factor-1. Despite stimulating proliferation, keratinocyte, fibroblast, or insulin-like growth factors did not stimulate directed migration. Chemotaxis required tyrosine kinase and phosphatidylinositolphospholipase C activities but not protein kinase C ormitogen-activated protein kinase activity. These findings suggest thatthe repertoire of growth factors capable of regulating directedintestinal epithelial cell migration is limited and that a divergenceexists in the signal transduction pathways for directed vs. nondirected migration.

     

Establishment and cellular characteristics of a hepatocyte cell line (oums-31) derived from an acatalasemic mouse

Summary Liver cell lines with very low catalase activity were established from an acatalasemic mouse. Hepatocytes isolated by a collagenase-liver-perfusion technique were cultured in Williams’ E medium supplemented with 10% fetal bovine serum. The acatalasemic liver cell line showed approximately 20% of the catalase activity of a normal mouse liver cell line, whereas its glutathione peroxidase activity was approximately equal to that of the normal liver cell line. DNA sequence analysis of this cell line showed the same mutation in the catalase gene as is seen in the acatalasemic mouse. Our observation of intracellular content of hydrogen peroxide (H₂O₂) radical and increased susceptibility of the cells to H₂O₂ were compatible with the existence of low catalase activity in the acatalasemic mouse. This hepatocyte cell line should be useful for studying effects of oxidative radical stress at the cellular level.     

Amphiregulin mediates self-renewal in an immortal mammary epithelial cell line with stem cell characteristics

Amphiregulin (AREG), a ligand for epidermal growth factor receptor, is required for mammary gland ductal morphogenesis and mediates estrogen actions in vivo, emerging as an essential growth factor during mammary gland growth and differentiation. The COMMA-D β-geo (CDβgeo) mouse mammary cell line displays characteristics of normal mammary progenitor cells including the ability to regenerate a mammary gland when transplanted into the cleared fat pad of a juvenile mouse, nuclear label retention, and the capacity to form anchorage-independent mammospheres. We demonstrate that AREG is essential for formation of floating mammospheres by CDβgeo cells and that the mitogen activated protein kinase signaling pathway is involved in AREG-mediated mammosphere formation. Addition of exogenous AREG promotes mammosphere formation in cells where AREG expression is knocked down by siRNA and mammosphere formation by AREG^−/− mammary epithelial cells. AREG knockdown inhibits mammosphere formation by duct-limited mammary progenitor cells but not lobule-limited mammary progenitor cells. These data demonstrate AREG mediates the function of a subset of mammary progenitor cells in vitro.     

Modulation of human fetal hepatocyte survival and differentiation by interactions with a rat liver epithelial cell line

Our recent studies have shown that chick embryo epiphyseal cartilage synthesizes three distinct species of proteoglycan (PG-H, PG-Lb, and PG-Lt) which are analogous in having glycosaminoglycan side chains of the chondroitin (dermatan) sulfate type but different from one another in regard to the structure of core protein. In the present report, the expression of PG-H and PG-Lb has been studied in developing chick hind limbs (stages 19-33), using antibodies specific for these substances in indirect immunofluorescence. At the onset of cartilage morphogenesis (stage 24), PG-H became recognizable in the cartilage primordia, whereas a parallel section stained for PG-Lb showed no reaction. The first evidence of PG-Lb appearance was seen in a stage 28 cartilage (e.g., tibia) in which the cells in the middiaphysis became elongated in a direction perpendicular to the long axis of the cartilage. The PG-Lb fluorescence was confined to the zone of these flattened, disc-like cells, whereas the fluorescence for PG-H was uniformly distributed throughout the cartilage. With further development of cartilage (stage 29 approximately), the zone of flattened cells spread proximally and distally, and simultaneously large hypertrophied cells appeared at the diaphyseal region. During these zonal changes of cell morphology, the PG-Lb fluorescence remained restricted to the zone of flattened cells. Parallel sections stained for PG-H, in contrast, showed an evenly distributed pattern of the PG-H fluorescence throughout the cartilage. The results indicate that the appearance of PG-Lb is closely associated with the zonal changes of cell shape and orientation along the proximal-distal axis of the developing limb cartilage, and further suggest that the flattened chondrocytes in this particular zone have undergone additional changes in gene expression to form an extracellular matrix of still another chemical property.     

Growth-suppressive function of human connexin32 in a conditional immortalized mouse hepatocyte cell line

Mouse hepatocytes immortalized with a temperature-sensitive allele of the SV40 large T-antigen (CHST8 cells) were found to lack the high expression of the gap junction proteins Cx26 and Cx32 that characterizes normal mouse hepatocytes, expressing instead Cx43 and Cx45 at minimal levels. In order to examine the growth suppressive function of Cx32 on hepatocytes, we transfected these CHST8 cells with human Cx32 complementary deoxyribonucleic acid and measured the growth rates at 33, 37, and 39 degrees C. Expression of human Cx32 and its messenger ribonucleic acid in the stable cell lines was confirmed by immunocytochemistry and by Western and Northern blots analyses. Dye transfer following lucifer yellow injection into the transfectants was extensive; Cx32 channels displayed unitary conductances of about 70 pS and were moderately voltage sensitive. When cultured at 33 and 39 degrees C, growth rates of both parental cells and transfectants were of the same level. When examined at 37 degrees C, growth rate of the transfectant, which highly expressed Cx32 at the membranes, was significantly decreased compared to the parental cells. However, no changes in the expression of Cx32 protein in the transfectants were observed between 33 and 37 degrees C. These results suggest that Cx32 expression could inhibit hepatocyte growth in vitro using the conditional immortalized cells. Cx32 transfectants using a conditional immortalized mouse hepatocyte may be useful for examining the mechanisms of growth and differentiation in hepatocytes by gap junction expression.     

Establishment of a human malignant fibrous mesothelioma cell line and the biological characteristics compared with malignant epithelial mesothelioma cell line

Two human malignant mesothelioma cell lines, which we designated "epithelial mesothelioma cells" and "fibrous mesothelioma cells", were established from the pleural fluid containing malignant mesothelial cells of a 72-year-old Japanese man. These cell lines were separated by the colonial techniques from the initiation of the primary cultures and grew well without interruption for 12 years. They were characterized as producing hyaluronic acid. These cell lines displayed different biological characteristics, including morphology, heterotransplantability and genetics using with BAC array CGH. The epithelial mesothelioma cells were epithelial in shape and transplantable into the subcutis of nude mice, while the cells of the fibrous mesothelioma line were fibroblast-like and transplantable into the submucosa of Hamster's cheek pouches but not into the subcutis of nude mice. The mesotheliomas are classified into three types: epithelial mesothelioma, fibrous mesothelioma and mixed type. The gene copy number losses observed on 9p21.3, 9p21.2, 9p21.1, among others may be a major mechanism of malignant mesothelioma carcinogenesis. We considered and supported the combination theory for the histogenesis of malignant mesothelioma.     

Quantitative comparison of rat hepatocyte functions in two improved culture systems with or without rat liver epithelial cell line

We quantitatively evaluated two recently-developed novel techniques for hepatocyte cultivation in a dish level; that is, spheroid culture and membrane-supported collagen (CN) gel sandwich culture, in terms of cellular maintenance, albumin secretion and 7-ethoxycoumarin (7EC) metabolism to 7-hydroxycoumarin (7HC) as a marker for cytochrome P450 IA1 activity in the presence and absence of rat liver epithelial cell line (RLEC) during one month of culture, together with conventional coculture with RLEC in CN-coated dishes as a control. RLEC prevented spheroid loss caused by its detachment from the culture dishes often occurring in pure culture. CN-gel sandwich by itself improved remarkably hepatocyte maintenance when compared with CN-gel free systems, thereby resulting in enhancement of overall functional expressions as compared with CN-gel free systems. RLEC in CN-gel sandwhich, however, reduced cellular sustainment probably due to its suppression of hepatocyte growth. Although there were no significant differences in albumin secretion per cell among the five cultures examined, CN-gel sandwich expressed markedly higher 7EC metabolizing activity per cell, where RLEC presence had a preferable influence. Consequently, membrane-supported CN-gel sandwich was the most superior technique for hepatocyte cultivation from the standpont of both cellular maintenance and its functional expressions per cell.     

Calcium and vitamin D modulate mouse colon epithelial proliferation and growth characteristics of a human colon tumor cell line

The role of calcium and vitamin D in the prevention of colorectal cancer is only now being investigated at the organismal, cellular, and molecular biologic levels. Recent epidemiologic studies have supported the hypothesis that dietary calcium and vitamin D may be related to a reduced risk for colon cancer. The evidence from laboratory investigations in animals and in cell culture also indicate a possible preventative effect. Addition of calcium and vitamin D to the roster of developmental cancer chemopreventative agents for further research is warranted.     

Induction of thymocyte proliferation by supernatants from a mouse thymic epithelial cell line

The thymic stroma plays a critical role in the generation of T lymphocytes by direct cell-to-cell contacts as well as by secreting growth factors or hormones. The thymic epithelial cells, responsible for thymic hormone secretion, include morphologically and antigenically distinct subpopulations that may exert different roles in thymocyte maturation. The recent development of thymic epithelial cell lines provided an interesting model for studying thymic epithelial influences on T cell differentiation. Treating mouse thymocytes by supernatants from one of TEC line (IT-76M1), we observed an induction of thymocyte proliferation and an increase in the percentages of CD4-/CD8- thymocytes. This proliferation was largely inhibited when thymocytes were incubated with IT-76M1 supernatants together with an anti-thymulin monoclonal antibody, but could be enhanced by pretreating growing epithelial cells by triiodothyronine. We suggest that among the target cells for thymulin within the thymus, some putative precursors of early phenotype might be included.     

Turtle urinary bladder epithelial cell line

1. We compared enzymatic and functional properties of a turtle bladder cell line to those of turtle bladder epithelial cells. Like the original epithelium, the cell line displays carbonic anhydrase activity and acetazolamide inhibits O2 consumption in isolated cells and acidification by cells grown in monolayers. 2. Staining with acridine orange revealed the presence of cytoplasmic orange red vesicles which could be dissipated by NH4Cl or protonophores indicating that these vesicles represent areas of low pH. 3. Addition of ATP to cells permeabilized by digitonin led to reappearance of the red granules suggesting that acidification of the vesicles is mediated by H+-ATPase.     

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.     

小鼠胸腺上皮细胞系MTEC1产生30kDr MCP—1类趋化因子

A medullary type mouse thymic epithelial cell line MTEC1 can produce chemokines. Crude culture supernant of MTEC1 cells was concentrated by controlled-pore glass beads. One of these chemotactic proteins was isolated from MTEC1 supernatant and purified to homogeneity by heparin-Sepharose affinity chromatography, cation-exchange FPLC and Reverse Phase-HPLC. The chemotactic factor for both lymphocytes and monocytes was identified as a 30 kD protein by SDS-PAGE analysis under reducing conditions. After cleavage of the NH2-terminally blocked protein with formic acid, the amino acid sequence of the internal fragment was analyzed and found to be identical to the amino acid sequence of mouse MCP-1/JE. The protein was hence identified as a glycosylated MCP-1/JE-like chemokine secreted by thymic epithelial cells. The characterization of chemokines produced by thymic stromal cells will benefit the analysis of the mechanism of thymus homing and the finding of new chemokines.     

Effect of mouse thymic medullary epithelial cell line on thymocyte development and functional maturationin vivo

Thymic medullary type epithelial cell line (MTEC1), which expressed H-2D^d and Ia^d, was derived from BALB/c mouse. MTEC1 cells were introduced by intrathymic injection into irradiated H-2^b mice reconstituted with H-2^bxd F1bone marrow cells. Two months later, the injected MTEC1 cells were found to be still present in the recipient thymus. Splenocytes from chimeric mice, inin vitro functional assays, were analyzed to investigate whether the MTEC1 cellsin vivo could induce the production of H-2^d restricted antigen-specific T cells. The H-2^d restricted VSV-antigen specific proliferating and IL-2 producing T cells as well as H-2^d restricted influenza virus specific cytotoxic T cells were found in chimeric mice injected with MTEC1 cells, and these cells were shown to be tolerant to H-2^d selfantigen. On the contrary, H-2^d restricted antigen-specific and H-2^d self-antigen tolerant T cells were not shown in control mice injected with saline. These results suggest that intrathymically injected MTEC1 cells could induce T lineage cell development and functional maturation in the intact thymus. A hypothesis of “second thymic selection” in thymic medulla has been postulated and its implication discussed.     

New acceptor cell for transfected genomic DNA: oncogene transfer into a mouse mammary epithelial cell line.

A line of mouse mammary epithelial cells (NMuMG) has been characterized for its ability to be stably transfected with exogenous DNA. A transfection frequency of at least 1 cell per 1,000 was obtained with the pSV2neo plasmid. Several thousand G418-resistant NMuMG cell clones can easily be generated in cotransfection of genomic DNA and pSV2neo. The NMuMG cells were isolated from normal mammary glands and do not form malignant lesions when injected into nude mice. We have cotransfected NMuMG cells with pSV2neo and genomic DNA from the human EJ bladder carcinoma line, a cell line which contains an activated c-rasH oncogene. When a pool of 4,700 G418-resistant colonies was injected into nude mice, tumors were obtained. These tumors contain a transfected human rasH gene. Genomic DNA transfection into a line of mouse epithelial cells, in combination with the selection of stable transfectants and tumor induction in nude mice, can be used to screen human tumor DNA for the presence of activated oncogenes.     

Establishment of epithelial cell strains from normal adult mouse lung resembling a urethane-induced lung adenoma cell strain and a metastasizing mouse lung carcinoma cell line

Epithelial cell strains, designated NAL, have been established from normal lungs of adult female Balb/c mice. The ultrastructural characteristics, effect of dexamethasone on cellular morphology and proliferation rate, and the cytoskeletal protein pattern of NAL suggests that these cell strains may be related to a urethane-induced mouse lung adenoma cell strain NUL and to a metastasizing mouse lung tumour cell line CMT. NAL exhibited no anchorage-independent growth under normal conditions, however extensive passaging in the presence of 5 X 10(-6)M dexamethasone resulted in a colony forming efficiency in soft agar of 8.4% at passage number 30.     

Spontaneous establishment of a novel Japanese macaque cell line with epithelial cell phenotypes

A novel macaque cell line (J3K) with epithelial phenotypes was spontaneously established from a kidney specimen of a Japanese macaque (Macaca fuscata). Its population doubling level reached more than 500, and it was regarded as an established permanent cell line. J3K cells have transformed cell phenotypes such as loss of contact inhibition and anchorage-independent cell growth. Unlike other monkey adherent cell lines, J3K had no SV40 large T antigen. After establishment, cells have constantly expressed telomerase activity, whereas telomere length has been maintained. No mutations in the coding regions of the p53 complementary deoxyribonucleic acid were detected in the late-passaged cells. J3K, a novel transformed epithelial cell line, will be useful material for the comparative study of human and other primate cellular aging as well as cancer cell biology.