The Importance of the Canonical Wnt Signaling Pathway in the Porcine Endometrial Stromal Stem/Progenitor Cells: Implications for Regeneration.

The regenerative ability of the endometrium is strongly associated with the presence of adult stem/progenitor cells. Purposes of the present study were (1) to establish the presence of stem/progenitor cells in porcine endometrial stroma using a clonogenic assay and (2) to investigate whether the canonical Wnt pathway affects the potential of stem/progenitor cells to undergo self-renewal or differentiation. The utility of endometrial stromal clones as a model for stem/progenitor studies was evaluated based on these cells' increased expression of mesenchymal stem cell (MSC) marker genes, including CD29, CD73, CD90, and CD105, compared with primary cultured cells. Small molecules were introduced to activate (BIO) or inhibit (XAV939) the canonical Wnt pathway during stromal clone formation. Cloning efficiency assays revealed that activation of the Wnt/ß-catenin pathway promoted formation of more differentiated small clones. Moreover, activation of the Wnt/ß-catenin pathway decreased, whereas inhibition of the pathway increased MSC marker expression. Additionally, we confirmed the importance of canonical Wnt pathway stimulation in endometrial stromal cells through observing the appropriate changes in ß-catenin cellular localization. These data indicate that modulation of the canonical Wnt pathway effects the process of regeneration in the porcine endometrium during the course of the estrous cycle.

Altered expression of the PLAGL1 (ZAC1/LOT1) gene in colorectal cancer: Correlations to the clinicopathological parameters.

Pleomorphic adenoma gene-like 1 gene (PLAGL1) encodes a zinc-finger nuclear transcription factor which promotes apoptosis and cell cycle arrest. Loss or downregulation of its expression has been observed in various human neoplasms. This study compared PLAGL1 expression in colorectal cancer (CRC) tissue and colon mucosa of healthy subjects at the mRNA and protein levels, and estimated its prognostic value. The PLAGL1 mRNA levels were also determined in CRC cell lines. We collected paired tumor tissue and unchanged mucosa of the large intestine from 121 CRC patients as well as 72 colon biopsies of healthy subjects obtained during screening colonoscopy. PLAGL1 mRNA levels were determined by quantitative PCR, while PLAGL1 protein expression was estimated by western blotting and immunohistochemistry. PLAGL1 mRNA level in tumor tissue was ~2-fold lower than in samples of corresponding unchanged tissues and biopsies of healthy colon mucosa. Downregulated expression of PLAGL1 mRNA was also observed in all tested CRC cell lines. Although the average content of PLAGL1 protein did not differ significantly between tumor and unchanged tissues of CRC patients or colon mucosa of healthy individuals, the decreased PLAGL1 protein levels in tumor specimens correlated with lymph node involvement, the presence of metastases and higher TNM disease stage. The PLAGL1 expression level did not correlate significantly with patient overall survival; however, the hazard ratio for patients whose tumor tissues showed reduced PLAGL1 immunohistochemical staining was twice higher than in patients with increased PLAGL1 immunoreactivity. In conclusion, these results suggest that dysregulation of PLAGL1 expression may be involved to some extent in the progression of CRC, but the so far collected patient survival data do not confirm applicability of the PLAGL1 expression level as a prognostic factor in CRC.

Evidence for the presence of stem/progenitor cells in porcine endometrium.

A population of adult stem cells responsible for cyclic reconstructing and remodeling has been proposed to reside in the highly regenerative mammalian endometrium. Recently, stem/progenitor cells have been identified in the human and mouse endometrium, but less is known about these cells in livestock animals. Using Hoechst 33342 fluorescent dye staining and flow cytometry, we identified an emerging cell side population that may be responsible for the regeneration process of the porcine endometrium. The percentage of side-population cells on Day 19 of the estrous cycle was significantly higher than that on Days 2-4. Moreover, single cells were able to seed clones that could differentiate into three independent mesenchymal-cell lineages. We also demonstrated the expression of specific markers of self-renewal cells on these side-population cells and the presence of a population of cells among the stromal cells that possess markers for mesenchymal stem cells. These results indicate that the porcine endometrium contains a population of cells with the capacity for self-renewal and a high rate of proliferation, which depend on the phase of the estrous cycle. These cells could potentially be involved in the cyclic reconstruction of the porcine endometrium.

Identification of pluripotent cells in bovine uterus: in situ and in vitro studies.

The aim of this study was to identify uterine pluripotent cells both in bovine uterine tissues as well in epithelial, stromal, and myometrial uterine cell populations. Moreover, the relationship of pluripotent markers expression with age and the uterine horn side was considered. Uterine tissue was collected from ipsilateral and contralateral horns (days 8-10 of the estrous cycle). Immunohistostaining for C-KIT, OCT3/4, NANOG, and SOX2 in uterine tissue was determined. mRNA expression of C-KIT, OCT3/4, NANOG and SOX2 was evaluated in uterine tissue relative to the age of the cow and uterine horn side. Gene and protein expression of these markers in the uterine luminal epithelial, stromal, and myometrial cells was evaluated by real-time PCR and western blotting respectively. The expression of pluripotent cell markers OCT3/4, NANOG, and SOX2 was identified by flow cytometry assay in epithelial, stromal, and myometrial cells. Multilineage differentiation of the bovine uterine cells was performed. mRNA expression of OCT3/4, NANOG, and SOX2 in uterine tissue was higher in the ipsilateral horn than in the contralateral horn. Flow cytometry assay revealed positive fluorescence for OCT3/4, NANOG, and SOX2 in all uterine cell types. Results showed the age-dependent expression of pluripotent markers in uterine tissue. Beside, the different expression of pluripotent cells in each horn of uterus suggests the influence of ovarian hormones on these characteristics. The highest mRNA and protein expression for pluripotent markers was observed in stromal cells among uterine cells, which indicates this population of cells as the main site of pluripotent cells in the cow uterus.

Effects of seminal plasma and the presence of a conceptus on regulation of lymphocyte-cytokine network in porcine endometrium.

Infusion of seminal plasma in the uterus is known to elicit an instant inflammatory response in the porcine uterus, but whether or not it prepares a uterine immunological response to the presence of conceptuses is not well understood. Seminal plasma induced long-term modulatory effects and conceptus-induced immune changes in leukocyte populations were measured by flow cytometry and mRNAs for various cytokines by quantitative reverse-transcriptase PCR in porcine endometrium collected on Days 6 and 13 from cycling and pregnant animals or from animals given seminal plasma infusions. Seminal plasma infusion induced long-term modulatory effects, resulting in significantly more endometrial FoxP3-positive T-regulatory and T-helper cells 6 days after infusion as compared to cycling and pregnant animals. The number of T-cytotoxic and T-null cells did not change between the studied groups. The early molecular effects of seminal plasma were not observed at 13-days post-infusion, although animals on Day 13 of pregnancy did show significantly more T-cells (of any type investigated). Seminal plasma also showed a delayed effect on cytokine expression, specifically exhibiting a significant increase in interleukin 10 (IL10) and a decrease in granulocyte macrophage colony-stimulating factor (GMCSF) gene expression on Day 13 as compared to Day 6 of cycling or pregnant gilts. The results indicate a delayed regulatory effect of seminal plasma on immune responses in the porcine uterus, which are similar to immune changes generated by implanting conceptuses.

Immortalization of swine umbilical vein endothelial cells (SUVECs) with the simian virus 40 large-T antigen.

Implementation of the swine umbilical vein endothelial cells (SUVECs) model in vitro can be instrumental in determining the biology of endothelial cells. We have generated an immortalized endothelial cell line, G-1410, using Simian virus 40 T-antigen (SV40 T-ag) primarily to overcome the short life span before the onset of senescence and high variability among enzymatically isolated cells of primary cultures. Fast proliferating cells were selected from cultures and, after a fifth passage, examined for the presence of the SV40 T-ag by PCR and immunocytochemistry. Phase contrast and transmission electron microscopy revealed that G-1410 cells did not differ morphologically from SUVECs. The G-1410 cells exhibited positive staining for vascular endothelial (VE)-cadherin and von Willebrand factor (vWF), and formed capillary-like tube structures on Matrigel. Despite the strong oncogenic signal provided by SV40 T-ag, these transformed G-1410 cells have remained karyotypically normal and non-tumorigenic. G-1410 cells also responded to stimulation with VEGF, FGF-2, and newborn calf serum. Moreover, G-1410 cells showed elevated expression of VEGF120, VEGF164 (VEGF-A), and FGF-2 at both mRNA and protein levels. In conclusion, based on the cytological and functional evaluation of the newly obtained immortalized cell line, it can be concluded that G-1410 cells provide a useful tool for studying the effects of VEGF and FGF systems, and other signal transduction pathways related to angiogenesis.

Characterization of bovine immortalized luteal endothelial cells: action of cytokines on production and content of arachidonic acid metabolites.

BACKGROUND: The interactions between luteal, vascular endothelial, immune cells and its products: steroids, peptide hormones, prostaglandins (PGs), growth factors and cytokines play a pivotal role in the regulation of corpus luteum (CL) function. Luteal endothelial cells undergo many dynamic morphological changes and their action is regulated by cytokines. The aims are: (1) to establish in vitro model for bovine luteal endothelial cells examination; (2) to study the effect of cytokines: tumor necrosis factor alpha (TNFalpha) and interferon gamma (IFNgamma) on cell viability, leukotrienes (LTs) and PG synthases, and endothelin-1 (EDN-1) mRNA, protein expression and their secretion in bovine immortalized luteal endothelial (EnCL-1) cells. METHODS: The primary cultures of bovine luteal endothelial cells were immortalized by transfection with vector carrying the Simian virus 40 T-antigen (SV40 T-ag) sequence. Expression of SV40 T-ag gene in EnCL-1 cells was confirmed by RT-PCR and immunofluorescence staining showed the presence of endothelial cell markers: VE-cadherin and von Willebrand factor. EnCL-1 cells were stimulated by TNFalpha with IFNgamma (50 ng/ml each) for 24 h. Cell viability, mRNA expression (real time RT-PCR), protein expression (western blotting) for LTC4 synthase (LTC4S), LTA4 hydrolase (LTA4H), PGE2 and PGF2alpha synthases and endothelin-1 (EDN-1), and levels of LTs (B4 and C4) and PGs (E2 and F2alpha) and EDN-1 in the medium (EIA) were evaluated. RESULTS: We received immortalized luteal endothelial cell line (EnCL-1). Cytokines did not change EnCL-1 cell viability but increased mRNA expression of LTC4S, LTA4H, PGE2 and PGF2alpha synthases and EDN-1. EDN-1/2/3, LTC4 and PGF2alpha synthases protein expression were elevated in the presence of TNFalpha/IFNgamma, and accompanied by increased EDN-1, LTC4 and PGF2alpha secretion. Cytokines had no effect on PGES and LTA4H protein expression, and PGE2 and LTB4 release. CONCLUSIONS: TNFalpha and IFNgamma modulate EnCL-1 cell function. Moreover, established EnCL-1 cell line appears to be a good model for investigating the molecular mechanisms related to cytokines action and aa metabolites production in cattle.

Assessment of VEGF-receptor system expression in the porcine endometrial stromal cells in response to insulin-like growth factor-I, relaxin, oxytocin and prostaglandin E2.

Several factors participate in regulation of growth and development as well as angiogenesis of the uterus during pregnancy, and hence little is known about the role of hormonal regulation of vascular endothelial growth factor (VEGF)-receptor system expression. This study has examined the effect of insulin-like growth factor-I (IGF-I), relaxin (RLX), oxytocin (OT) and prostaglandin (PG) E(2), on VEGF secretion and VEGF-receptor system mRNA expression in the porcine endometrial stromal cells. IGF-I and RLX were identified as the most effective inducers of VEGF secretion and mRNA expression. Although PGE(2) stimulated VEGF secretion and VEGF164 mRNA expression, OT inhibited both secretion and mRNA expression of VEGF. When tested for VEGF receptors (R), all factors failed to affect their mRNA expression. Media conditioned by stromal cells collected after IGF-I and RLX treatment significantly increased endothelial cell proliferation and this effect was blocked by soluble VEGFR-1. These data suggest that during early pregnancy IGF-I, RLX and PGE(2) can affect VEGF expression in the endometrium and therefore may support uterine and embryo development, implantation and pregnancy.

A novel targeted therapy of Leydig and granulosa cell tumors through the luteinizing hormone receptor using a hecate-chorionic gonadotropin beta conjugate in transgenic mice.

We investigated the antitumoral efficacy, endocrine consequences, and molecular mechanisms underlying cell death induced by the Hecate-chorionic gonadotropin (CG)beta conjugate, a fusion protein of a 23-amino acid lytic peptide Hecate with a 15-amino acid (81-95) fragment of the human CGbeta chain. Transgenic (TG) mice expressing the inhibin alpha-subunit promoter (inhalpha)/Simian Virus 40 T-antigen (Tag) transgene, developing luteinizing hormone (LH) receptor (R) expressing Leydig and granulosa cell tumors, and wild-type control littermates were treated either with vehicle, Hecate, or Hecate-CGbeta conjugate for 3 weeks. Hecate-CGbeta conjugate treatment reduced the testicular and ovarian tumor burden (P < .05), whereas a concomitant increase (testis; P < .05) or no change (ovary) in tumor volumes occured with Hectate treatment. A drop in serum progesterone, produced by the tumors, and an increase in LH levels occured in Hecate-CGbeta treated mice, in comparison with vehicle and Hecate groups, providing further support for the positive treatment response. Hecate-CGbeta conjugate induced a rapid and cell-specific membrane permeabilization of LHR-expressing cells in vitro, suggesting a necrotic mode of cell death without activation of apoptosis. These results prove the principle that the Hecate-CGbeta conjugate provides a novel specific lead into gonadal somatic cell cancer therapy by targeted destruction of LHR-expressing tumor cells.

Targeted ablation of prostate carcinoma cells through LH receptor using Hecate-CGbeta conjugate: functional characteristic and molecular mechanism of cell death pathway.

A Hecate-CGbeta conjugate (lytic peptide and beta-chorionic gonadotropin) selectively destroyed cells possessing LH receptors. This study described functional characteristics of the conjugate and the molecular mechanism of the cell death pathway in prostate cancer cells. Based on in vitro studies, we conclude that the conjugate kills cells possessing luteinizing hormone receptors (LHR) faster than Hecate alone. Competitive studies have shown that blocking of LHR by preincubation with chorionic gonadotropin (100 ng/ml) reduced toxicity of the conjugate in low concentrations. Further studies have also shown that the conjugate in treated cells both did not induce internucleosomal DNA fragmentation and did not induce morphological changes in cells characterized as having apoptotic features. These results proved that cells died by necrosis rather than apoptosis after the conjugate treatment.

Growth repression in diethylstilbestrol/dimethylbenz[a]anthracene-induced rat mammary gland tumor using Hecate-CGbeta conjugate.

Recently, we have shown that Hecate-CGbeta conjugate, which is a fusion of the lytic peptide Hecate and a 15-amino acid fragment of the beta-chain of chorionic gonadotropin (CGbeta), selectively destroys mammary gland carcinoma cells that possess luteinizing hormone receptors (LHR) in vitro. We induced mammary gland tumors using combined prenatal exposure to synthetic diethylstilbestrol (DES) and additional postnatal exposure to dimethylbenz[a]anthracene (DMBA). Rats with tumors were equally randomized (10/group) and treated with either sham (control) or 12 mg/kg body wt of either Hecate or Hecate-CGbeta once a week for 3 weeks by tail vein injections. One week after the last injection, rats were killed. Reverse-transcription-nested polymerase chain reaction/Southern blotting revealed alternatively spliced mRNA for LHR in tumor tissues of 5 of 30 females, which was further confirmed by Western blot analysis. The percentage of tumor volume increase was lowest in the group treated with Hecate-CGbeta (45.3 +/- 27.6), compared with Hecate- and sham-treated, control group (324.8 +/- 78.1 and 309.9 +/- 51.2, respectively; P<0.001). Hecate-CGbeta induced a significant decrease in tumor burden compared with controls (9.5 +/- 2.1 mg/g body wt vs. 21.6 +/- 2.9; P<0.01). A smaller reduction in tumor burden was also observed in Hecate-treated females (17.6 +/- 1.6 mg/g body wt vs. 21.6 +/- 2.9; P<0.05). Our results prove the principle that Hecate-CGbeta conjugate is able to repress mammary gland tumor growth, even in tumor tissues that lack or have very low levels of LHR. The mechanism of Hecate-CGbeta conjugate action in repression of DES/DMBA-induced tumor growth needs to be further analyzed to clarify the molecular mechanisms of Hecate-CGbeta conjugate action in vivo.

A novel approach of targeted ablation of mammary carcinoma cells through luteinizing hormone receptors using Hecate-CGbeta conjugate.

Recent studies have shown that human and animal mammary gland carcinoma cell line express luteinizing hormone receptors (LHRs). We have examined the cytotoxic effect of Hecate-CGbeta conjugate, that is, fusion of a lytic peptide (Hecate) and a 15-amino acid fragment of the CGbeta-chain in vitro. To test the hypothesis that the Hecate-CGbeta conjugate selectively abolishes cells possessing LHR, estrogen dependent and independent human breast cancer cell lines (MCF-7; MDA-MB-231) and a mouse Leydig tumor cell line (BLT-1) were treated in vitro with Hecate-CGbeta conjugate and Hecate alone. Cytotoxic effects of the Hecate-CGbeta conjugate and the Hecate alone was measured by lactate dehydrogenase (LDH) release immediately after treatment. We observed that the Hecate-CGbeta conjugate selectively, in dose-dependent manner destroys cells possessing LHR in lower concentrations of preparate comparing to the Hecate alone and that the cytotoxic effect is strongly correlated with the number of LHR. Using Western blot analysis we characterized the LHR on membranes of MDA-MB-231, MCF-7 and BLT-1 tumor cell lines. In addition, we showed the evaluation of inhibition potential of the Hecate-CGbeta conjugate to LHR. At a concentration of 33 microM the conjugate inhibited (50%; IC50) the binding of CG to LHR. We suggest further development of this novel approach for the treatment of breast cancer by the Hecate-CGbeta for in vivo trials.