Establishment of an androgen-responsive prostatic cell line "PEA5" from a p53-deficient mouse.

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 microg/ml), transferrin (10 microg/ml), cholera toxin (10 ng/ml) and selenium (10(-8) M). In the present study, 5alpha-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.

Indomethacin suppresses the growth of colon 26, Meth-A and FM3A tumors in mice by reducing the prostaglandin E2 content and telomerase activity in tumor tissues.

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.

Effectiveness of indomethacin as an antitumor agent in Colon 26-bearing conventional and nude mice, and telomerase activity in the tumors.

The antitumor effect of indomethacin on Colon 26 tumor was investigated in conventional (CDF1) and nude mice (BALB/c nu/nu), and the telomerase activity in the tumor tissues treated with indomethacin was monitored. Growth of Colon 26 tumor was significantly suppressed with indomethacin treatment compared to the controls both in conventional and nude mice. And telomerase activity in the tumor tissues noticeably declined in contrast to normal somatic tissues (testis, liver and colon), which were not affected by indomethacin treatment. We also showed that indomethacin can suppress tumor growth in association with a preferential decrease in telomerase activity in tumor tissues both in conventional and nude mice to the same extent. This study suggests a method for investigating the mechanism of tumor suppression by indomethacin, and suggests that indomethacin might be useful as a novel agent for human cancer therapy.

Establishment of prostatic cell line "Pro9ad" from a p53-deficient mouse.

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 microg/ml), transferrin (10 microg/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 5alpha-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.

The change in tenascin expression in mouse uterus during early pregnancy.

PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: These results indicate that TN expression in the uterus during early pregnancy is spatiotemporally different and may be regulated by a different mechanism.

The cDNA sequence of porcine vitronectin and its expression in liver and skeletal muscle of GH-supplemented pigs.

A cDNA (1555 bp) (DNA database accession number, D61396) having a homology with human vitronectin (Vn) was isolated from a porcine liver cDNA library, and its sequence was determined. The open reading frame in the cDNA was found to code a protein with 388 amino acids, then the amino acid sequence of the protein (porcine putative Vn) was aligned to the other mammalian (mouse, rabbit, and human) Vns previously reported. The alignment revealed that the functional amino acid sequences reported as the cell attachment site, the heparin binding site, the region involved in glycosylation, and plasminogen activator inhibitor I-binding domain were conserved in the porcine putative Vn. These findings together with the fact that the calculated molecular weight and the N-terminal amino acid sequence of the putative Vn agreed with those reported by biochemical analysis on porcine Vn, led us to conclude that the cDNA isolated in the present study coded for the porcine Vn. Then, a time course study was performed to examine whether the administration of growth hormone (GH) affects Vn mRNA expression in liver and skeletal muscle, since the level of Vn mRNA was reported to be affected by inflammation, and since GH was reported to be involved in inflammation. This revealed that GH has no effect on the level of liver Vn mRNA, and that Vn mRNA level in skeletal muscle seemed to be affected following GH-injection.

Developmental analysis of the eye lens obsolescence (Elo) gene in the mouse: cell proliferation and Elo gene expression in the aggregation chimera.

This study investigates the primary effect of the eye lens obsolescence (Elo) gene of the mouse. Morphological features of the Elo lens were defined as follows: (1) deficient elongation of lens fiber cells, (2) morphological abnormality of nuclei of lens fiber cells, (3) lack of eosinophilic granules in the central fiber cells and (4) rupture of lens capsule in the posterior region. We have immunohistologically examined, by means of an in vivo BrdU incorporation system, whether or not the Elo gene regulates cell proliferation during lens development. The lens fiber cells were morphologically abnormal in day 13 embryonic Elo lens. However, there were no significant differences in morphology or cell proliferation between normal and Elo lens epithelium until day 14 of gestation. After day 15, the total cell number in the Elo lens epithelium was significantly less than that in the normal, but the total numbers of S-phase cells in the two genotypes were not significantly different. The ratio of the total S-phase cell number to the total number of lens epithelial cells may be affected by the developmental stage, but not directly by the genotype. The genotype, however, may be having a direct influence at later ages because malformation of Elo lens fiber cells must cause reduction of the total number of lens epithelial cells in older embryos. Although, at 30 days old, Elo lens cells were externally extruded through the ruptured capsule into the vitreous cavity, BrdU-labelled lens epithelial cells were detectable. To investigate whether the Elo lens phenotype is determined by its own genotype or by its cellular environment, we produced aggregation chimeras between C3H-Elo/+(C/C) and BALB/c(c/c). Most lenses of BALB/c dominant chimeras were oval in shape without the ruptured lens capsule. However, they were opaque in the center and slightly smaller in size than normal. The lenses of C3H-Elo/+ dominant chimeras were morphologically similar to the Elo lens. Although normal nuclei were regularly arranged in the anterior region, Elo-type nuclei were located in the posterior region. Immunohistological staining by using anti-C3H strain-specific antibody demonstrated that the lens fiber cells with abnormal nuclei were derived only from C3H-Elo/+, not from BALB/c. These observations suggest that the primary effect of the Elo gene in the developing lens may be specific to the fiber cell differentiation rather than to the cell proliferation.(ABSTRACT TRUNCATED AT 400 WORDS)

DNA replication in uterine cells of adult and prepubertal mice under normal and hormonally stimulated conditions detected by bromodeoxyuridine labeling method.

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.

Immunohistochemical detection of DNA replication in mouse uterine cells by bromodeoxyuridine labeling of wax- and resin-embedded tissue sections.

To apply the bromodeoxyuridine (BrdU) labeling method using a monoclonal antibody to the study of cell proliferation in the mouse uterus, methods of fixation and embedding of tissues and of immunofluorescent staining were compared in terms of the rate of detection of labeled cells and specificity and stability of fluorescence obtained. BrdU was administered intravenously 2 hr before death and uterine blocks were embedded in polyester wax and Technovit resin after fixation in formalin and periodate-lysine-paraformaldehyde, respectively. The indirect method with anti-BrdU and fluorescein isothiocyanate (FITC) conjugated antimouse IgG antisera and the direct method with FITC conjugated anti-BrdU antibody were applied to both wax- and resin-embedded sections. Labeled and total cells were counted in luminal and glandular epithelia and stomata adjoining them. Counterstaining with hematoxylin for counting total cells produced intense fluorescence over the whole of resin sections and made counting of labeled cells impossible. On wax sections, on the other hand, the results were satisfactory, although the number of labeled cells detected was decreased slightly. In wax sections fluorescence due to nuclear incorporation of BrdU in the indirect method could be easily distinguished from the cytoplasmic or extracellular emission seen in some cells by its location and characteristic color. In resin sections, however, more careful observation was needed since the second antibody used in the indirect method cross-reacted with IgG in eosinophils and produced cytoplasmic fluorescence of the same color. By the indirect method greater numbers of labeled cells were detected in wax sections than in resin sections. The difference was distinct in tissues with extensive cell proliferation. By the direct method the fluorescence obtained was weaker and apt to fade more quickly than that obtained by the indirect method; use of the direct method reduced the number of labeled cells detected in both wax- and resin-embedded sections.