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The antitumor effect of indomethacin on Colon 26, Meth-A and FM3A tumors was investigated in mice. The prostaglandin E2 content in tumor tissues was assayed to find out if indomethacin acts on tumors, and the telomerase activity in tumors and somatic tissues (testis, liver, spleen and colon) was also monitored during indomethacin treatment. Growth of Colon 26, Meth-A and FM3A tumors was significantly (P < 0.001-0.05) suppressed by indomethacin compared to the untreated controls. The prostaglandin E2 content in the three tumors was markedly (P < 0.001) reduced by indomethacin. Telomerase activity in Colon 26 and FM3A tumors was significantly (P < 0.001) lower than that of untreated tumors (80% and 45% decrease versus the controls, respectively), and the activity in Meth-A tumor was slightly decreased (10% decrease versus the control) by indomethacin. Telomerase activity in the somatic tissues was not significantly affected by indomethacin. In summary, this study shows the effectiveness of indomethacin as an antitumor agent against three types of tumors, and suggests that indomethacin affects telomerase activity in tumors in vivo.

UE8 is a uterine epithelial cell line established from p53-deficient fetal female mice. UE8 exhibits a typical epithelial morphology in culture and is strongly positive for cytokeratin, but negative for vimentin in immunocytochemistry. UE8 shows an active growth in a phenol red free 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's nutrient mixture F-12 supplemented with 3% heat-inactivated and dextran-coated charcoal-treated fetal calf serum. Both immunocytochemistry and immunoblot analyses confirmed that UE8 was negative for the estrogen receptor. Diethylstilbestrol added to the medium at concentrations between 10−6 and 10−8 M had no significant effect on the proliferative rate, and estradiol-17β at 10−6 M was slightly inhibitory. Unexpectedly, however, the“pure anti-estrogen”ICI 182,780 at 10−6 M significantly enhanced cell proliferation. This is the first observation that the“pure anti-estrogen”ICI 182,780 stimulates the growth of uterine epithelial cells without estrogen receptors.

BACKGROUND We demonstrated that p53-deficiency is sufficient for immortalization of fetal uterine cells. In the present study, we further extended our previous observations to prostate tissues from a young p53-deficient adult mouse. METHODS Cell lines were established from the ventral prostate of a p53-deficient male mouse and maintained in medium containing 10% heat-inactivated fetal calf serum supplemented with insulin (10 μg/ml), transferrin (10 μg/ml), cholera toxin (10 ng/ml), and selenium (10−8 M). RESULTS Pro9ad, one of the lines established, exhibits a typical epithelial morphology in culture. Despite the possession of androgen receptors, the growth of Pro9ad was not stimulated by 5α-dihydrotestosterone. Hepatocyte growth factor (HGF) slightly stimulated proliferation, whereas fibroblast growth factor-1 (FGF-1), keratinocyte growth factor (KGF), and platelet-derived growth factor AB (PDGF-AB) had no stimulating effect on growth. However, FGF-2, epidermal growth factor (EGF), and insulin-like growth factor-1 (IGF-1) accelerated proliferation in a dose-dependent manner. EGF and IGF-1 additively stimulated growth. CONCLUSIONS These results suggest that Pro9ad shares characteristics in common with primary prostatic epithelial cells despite p53-deficiency, and that p53-deficiency alone allows establishment of clonal cell lines of the prostate epithelium. Furthermore, the prostates of p53-deficient mice are useful sources for obtaining cell lines. Prostate 36:102–109, 1998. © 1998 Wiley-Liss, Inc.

Background This study uses Colon 26-bearing CDF1 mice to assess the pharmacologic actions of indomethacin on tumor growth and cancer cachexia. The effect on tumor growth was evaluated by tumor weight, and the effect on cachexia was screened by changes in body weight and food intake. Additional indices measured included serum levels of immunosuppressive acidic protein and interleukin-6, and prostaglandin E2 content in the tumor tissues.

UE8 is one of several uterine cell lines established from p53-deficient fetal female mice. UE8 exhibits a typical epithelial morphology in culture; it is strongly positive for cytokeratin, but negative for vimentin. Immunoblot analysis confirmed that UE8 has glucocorticoid receptors. It grows actively in medium supplemented with 3% heat-inactivated, dextran-coated charcoal-treated fetal calf serum. Dexamethazone (DEX) inhibited its growth dose-dependently at concentrations between 10−6and 10−9 M. The inhibition by DEX was further examined in chemically defined medium (CDM): DMEM/F12 containing transferrin (10 μg/ml), selenium (10−8 M) and 0.1% bovine serum albumin. DEX at 10−6 M inhibited the growth of UE8 in CDM or in CDM supplemented with insulin-like growth factor-1 (IGF-1: 10 ng/ml), but there was no inhibition by DEX(10−6M) in the presence of epidermal growth factor (10 ng/ml). Thus, DEX inhibits the growth of a uterine epithelial cell line derived from p53-deficient mice in vitro, and also ...

Completely everted uterine horns of proestrous mice were treated with trypsin at 4°C and with a minimum mechanical agitation, and their surface epithelia were separated from the stroma and uterine glands with a minimum contamination of non-epithelial cells. The epithelial cell fraction was further purified by collagenase-dispase treatment, Percoll gradient separation and short term cultures on the collagen gel (5 min×3). Immunocytochemical check of cytokeratin networks in the cytoplasm proved well the purity of the cell fraction finally obtained.The epithelial cells could proliferate well in the medium composed of a 1:1 mixture of Ham's F12 and Dulbecco's modified Eagle medium containing 50 μM of Ca2+ and supplemented with epidermal growth factor (EGF), insulin, transferrin, bovine serum albumin (BSA), vitamin A and hydrocortisone, when seeded on the collagen gel at a density higher than 2×105/well (ca. 1, 000 cells/mm2). Deletion of anyone of insulin, transferrin and BSA from the culture medium suppressed severely the cell proliferation, while an only partial suppression was caused by the omission of EGF. Either single or simultaneous removal of vitamin A and hydrocortisone did not affect the proliferation at all. No stimulating effect of estradiol-17β on the proliferation was observed.

Clonal cell lines have been established from vaginae of prepubertal female p53-/- mice. Because the mouse vagina has a dual origin (the cranial three-fifths derived from the Mullerian duct and the caudal two-fifths derived from the urogenital sinus), both parts were separately subjected to cloning. Sixteen epithelial and two fibroblastic cell lines established from the cranial three-fifths (Mullerian vagina group), and four epithelial and three fibroblastic cell lines were established from the caudal two-fifths (sinus vagina group). They were maintained in Dulbecco's modified Eagle medium and Ham's nutrient mixture F-12 containing 10% fetal calf serum and 17β-estradiol at 10−8 M. Two cell lines (one epithelial and one fibroblastic) were examined using soft agar assay, but no colonies were formed. The doubling time of cell lines was approaximately 24 h, and all of them divided more than 200 times without crisis, suggesting that they were immortalized. All epithelial cell lines expressed cytokeratin 8. However, the epithelial cell lines expressed cytokeratin 14 and cytokeratin 10 when exposed to medium containing different concentrations of Ca2+. Fibroblastic cell lines expressed vimentin. All epithelial and fibroblastic cell lines expressed estrogen receptor-α protein. This is the first successful establishment of clonal cell lines from the normal mouse vagina, and these lines may provide good models in vitro of the vagina for the study of the mechanism of estrogen action.

Background We demonstrated that p53-deficiency is sufficient for the establishment of clonal cell lines from the uterus and prostate. In the present study, we improved cloning methods to establish androgen-responsive cell lines. Methods In our previous study, a prostatic cell line was established from the ventral prostate of a p53-deficient mouse and was maintained in a medium containing heat-inactivated fetal calf serum at 10% supplemented with insulin (10 μg/ml), transferrin (10 μg/ml), cholera toxin (10 ng/ml) and selenium (10−8 M). In the present study, 5α-dihydrotestosterone (10−8 M) was added to the medium from the beginning of cloning procedures. Results We succeeded in the establishment of an androgen receptor positive prostatic cell line, designated PEA5. PEA5 cells exhibited a typical epithelial morphology in culture and growth was stimulated by androgens in a dose-dependent manner. In addition, they grew and formed three-dimensional structures in collagen gel, in which growth was also stimulated by androgen. Conclusions Although PEA5 lacks p53 gene, it still retains androgen sensitivity. In collagen gel culture, PEA5 cells can grow and form three-dimensional structures similar to those of the primary cultures reported previously. Furthermore, prostates of p53-deficient mice are shown to be useful sources for obtaining androgen-responsive cells lines. Prostate 46:214–225, 2001. © 2001 Wiley-Liss, Inc.

Cervicovaginal secretions (CVSs) contain cytokines of maternal origin (1)(2), inflammatory cells, and proteolytic enzymes derived from leukocytes (3)(4) and act to protect the intraamniotic compartment from infectious agents that may ascend along the birth canal. CVSs have been found to contain ceruloplasmin (Cp) (5), a protein formed predominantly in the liver. The potential functions of Cp have been divided into two categories: (a) transportation of copper to tissue sites, and (b) functions such as oxidase activity of aromatic amines and serum antioxidation. In this setting, Cp acts as an extracellular scavenger of free radicals and superoxide ions and endogenously modulates inflammatory responses (6). The former function uses inactive Cp, and the latter function is carried out by active Cp. Hiyamuta et al. (7), using an original ELISA method that they established, found both inactive and active Cp in human serum. In a preliminary screening, active Cp was shown to be present in several external body secretions, such as in tears, salivary excretions, and nasal discharge. In this study, we used this assay system to measure active Cp in CVS in term pregnancy, and we found that increased active Cp in CVS was associated with premature rupture of the membranes (PROM). We enrolled 80 women at term pregnancy. With an informed consent from these 80 women, CVS was obtained from the cervical canal at ∼36 weeks of pregnancy, using a pair of sterilized cotton swabs (Japan Medical Service) at the time of regular antepartum checking. The total volume of collected CVS was weighed, and the mean CVS collected was ∼20 mg. One of the paired sterilized cotton swabs was used routinely for microbial culture. The other sterilized cotton swab was transferred to a silicon-coated polypropylene tube containing phosphate-buffered saline (1.0 mL) and rinsed vigorously. These samples were …

Our aim was to examine the changes in spatiotemporal tenascin (TN) expression in mouse uterus during early pregnancy, when the uterine tissue undergoes a tremendous restructuring.Using immunohistochemistry and in situ hybridization, the changes in distribution of TN protein in mouse uterine tissues in pregnancy Day 0 through Day 5 were analyzed.Immunoreactive TN and TN mRNA were expressed in the basement membrane of the epithelium as well as in the smooth muscle layer, and their distribution shifted from the subbasement region on Day 0-3 to the smooth muscle layer on Days 4 and 5.These results indicate that TN expression in the uterus during early pregnancy is spatiotemporally different and may be regulated by a different mechanism.

Oviductal functions have been studied mainly in primary epithelial cell culture and organ culture. However, secretory cells and ciliated cells coexist in the epithelium, and the small size of the oviduct limits the sources of both epithelial and stromal cells. To circumvent the limits, we attempted to establish clonal cell lines from an oviduct of a p53-deficient mouse. An oviduct was enzymatically digested and cultured in medium containing 10% fetal calf serum supplemented with estradiol-17beta. Morphologically distinct clones (10 epithelial and 4 fibroblastic clones) were established, and all clones expressed estrogen receptor alpha and progesterone receptor. Expression of a mouse oviduct-specific glycoprotein gene as a marker of secretory cells was limited in one clone and was stimulated by estrogens and suppressed by progesterone. Expression of helix factor hepatocyte nuclear factor/forkhead homologue-4 gene as a marker of ciliated cells was limited in two clones and was suppressed by estrogens. The two genes were never coexpressed in any clones. The results strongly suggest that the oviductal epithelium consists of two functionally determined populations. To our knowledge, this is the first establishment of functional clonal cell lines of the oviduct and makes it possible to study independently two oviductal functions, secretion and ciliogenesis.

To confirm the utility of the bromodeoxyuridine (BrdU) labeling method in the study of cell proliferation in mouse uterine tissues, changes in the labeling index in the luminal and glandular epithelia, the periluminal, periglandular and deep stromal regions and the myometrium were surveyed in normal adult mice during the estrous cycle and early pregnancy, in prepubertal mice and in ovariectomized adult and young animals treated with estrogen and/or progesterone. All results obtained were consistent with those obtained in previous histometric and autoradiographic studies and proved the effectiveness of the BrdU labeling method in the study of the uterus as well as many other organs. A marked rise in the labeling index was found in the luminal epithelium at metestrus, as well as on the proestrous morning, indicating the occurrence of extensive cell proliferation in the absence of estrogen stimulation. The change in the labeling index in adult mice was much more evident in the luminal epithelium than in the glandular epithelium in all conditions examined. On the other hand, the change in the stroma was more eminent in the periglandular region than in the periluminal and deep regions in most conditions. In immature mice, a great increase in labeling incidence occurred not only in luminal epithelium but also in muscle layers along with the process of puberty and at the time of estrogen stimulation. A moderate increase in the incidence also occurred in all other areas of the uterus including the perimetrium. Again, the increase was more prominent in the periglandular area than in other stromal regions.