Churl K. Min

Cloning and Expression of a G Protein-Linked Acetylcholine Receptor from Caenorhabditis elegans

Abstract : We have isolated a cDNA clone from the nematode Caenorhabditis elegans that encodes a protein of greatest sequence similarity to muscarinic acetylcholine receptors. This gene codes for a polypeptide of 682 amino acids containing seven putative transmembrane domains. The amino acid identities, excluding a highly variable middle portion of the third intracellular loop, to the human m1‐m5 receptors are 28‐34%. When this cloned receptor was coexpressed with a G protein‐gated inwardly rectifying K+ channel (GIRK1) in Xenopus oocyte, acetylcholine was able to elicit the GIRK current. This acetylcholine‐induced current was substantially inhibited by the muscarinic antagonist atropine in a reversible manner. However, another muscarinic agonist oxotremorine and antagonists scopolamine and pirenzepine had little or negligible effects on this receptor. Taken together, these results suggest that the cloned gene encodes a G protein‐linked acetylcholine receptor that is most similar to but pharmacologically distinct from muscarinic acetylcholine receptors.

A well-defined in vitro three-dimensional culture of human endometrium and its applicability to endometrial cancer invasion

A three-dimensional (3-D) endometrium culture was established, in which human endometrial stromal cells embedded in a mixture of collagen I, a major component of extracellular matrix, and matrigel, a basement membrane material, supports the epithelial cells seeded on top of the collagen/matrigel matrix. The biological growth and differentiation of the epithelial cells were studied microscopically and immunohistochemically. Transmission electron microscopy showed a polarized columnar epithelium in monolayer with basally positioned nuclei. Scanning electron microscopy revealed a confluent epithelium with an abundance of microvilli and cilia as well as pinopodes on the apical surface. An immunohistochemical staining showed that integrin alpha1, alpha4, and beta3 were co-localized with cytokeratin, confirming the epithelial origin of the cells. In contrast, immunoreactivity against cyclooxygenase-1 or -2 was positive in both epithelial and stromal cells. When epithelial cells were replaced by KLE cells, an endometrial cancer cell of epithelial origin, invasion of KLE cells into the stromal fraction was observed. The invasion was closely correlated to expression of matrix metalloproteinases and their tissue inhibitors of metalloproteinases in a manner consistent with paracrine fashion. The present 3-D culture imitates the normal endometrium physiologically as well as morphologically, thus provides an excellent in vitro tissue suitable for reproducing in vivo physiological processes, including endometrial cancer invasion.

Syndecan-1 enhances the endometrial cancer invasion by modulating matrix metalloproteinase-9 expression through nuclear factor κB

OBJECTIVES Up-regulated expression of syndecan-1, a member of the transmembranous proteoglycans that serves as a co-receptor for a wide pool of extracellular ligands, has been ascribed to the promotion of growth of various cancers including breast, ovarian, and endometrial cancers. Here, we have extended these observations to gain insight into correlation between the expression level of syndecan-1 and its tumor-promoting characteristics, particularly, cancer invasion, in endometrial cancer. METHODS Human syndecan-1 was stably transfected into three human endometrial cancer cell lines, and its effects were examined with respect to cell survival/proliferation and invasion. In addition, the activation of underlying signaling components, including integrins, focal adhesion kinase (FAK), and nuclear factor kappaB (NF-kappaB) was examined. The activity of NF-kappaB as a transcription factor for matrix metalloproteinase (MMP)-9 was assessed. RESULTS The innate expression level of syndecan-1 was moderate to high in all endometrial cancer cell lines. Overexpression of syndecan-1 promoted tumor cell proliferation concomitant with the activation of NF-kappaB. Furthermore, overexpression of syndecan-1 markedly enhanced the cancer invasion accompanied by enhanced expression of integrin alphav/beta5 and enhanced phosphorylation of FAK. The transcriptional activation of MMP-9 by NF-kappaB was up-regulated in syndecan-1 overexpression. CONCLUSION These findings provide evidence that supports that syndecan-1 may have a critical role in carcinogenic progression, particularly, contributing to the development of proliferative and invasive phenotype through NF-kappaB-mediated MMP-9 gene expression in endometrial cancer.

Syndecan-1, a key regulator of cell viability in endometrial cancer.

Syndecan‐1 is one of the major proteoglycans on cell surfaces involved in major biological processes. Although loss of syndecan‐1 correlates well with the gain of cancerous characteristics in a wide range of cancers, increased expression of syndecan‐1 also coincides with adverse outcomes in some cancers, including breast, ovarian and pancreatic cancers. For this Janus‐faced attitude of syndecan‐1, we sought to examine expression patterns of syndecan‐1 in endometrial carcinoma (EC) and gain insight into the roles of syndecan‐1. Immunohistochemical examinations of 109 endometrial tissue samples from myoma, hyperplasia and EC uteri revealed that syndecan‐1 expression was significantly upregulated in EC compared with hyperplasia (p < 0.001). To evaluate pathophysiological functions of syndecan‐1, its expression level was altered, and subsequent outcomes were examined using human endometrial cancer cell lines such as HEC‐1A, AN3CA and KLE cells. Overexpression of syndecan‐1 increased the growth of HEC‐1A cells regardless of anchorage dependence while silencing syndecan‐1 by antisense RNAs caused apoptotic cell death. Consistent with decreased viability, the loss of syndecan‐1 was also accompanied by a decrease in the activation of Erk and Akt and a concomitant decrease in the phosphorylation of PTEN and PDK1, which are known as negative and positive regulators of Akt activation, respectively. These down‐regulatory effects were reversed upon overexpression of syndecan‐1. Collectively together, the aforementioned findings lend support to the notion that upregulation of syndecan‐1 may be a critical element for endometrial cancers in maintaining their viability and thus can serve as a cancer specific therapeutic and diagnostic marker.

Endometrial cancer invasion depends on cancer-derived tumor necrosis factor-α and stromal derived hepatocyte growth factor

Cancer invasion is an outcome of interactions of the cancer and the host cell. It is now becoming increasingly clear that ovarian hormones have a huge influence on such intercommunications in various types of cancers. Estrogen is known to aggravate the aggressiveness of the endometrial cancer whereas progesterone seems to act as a negative factor. Insight into the mode of ovarian hormonal actions could come from the studies of its regulation of the paracrine interactions between the endometrial cancer and the normal stromal cells during the cancer invasion. In this context, we report here that estrogen promotes the endometrial cancer invasion by inducing humoral interactions between the cancer and the stromal cells, i.e., estrogen stimulates tumor necrosis factor‐α expression from the endometrial cancer cells, which, in turn, induces the stromal expression of hepatocyte growth factor (HGF), conferring the enhanced NK4 (HGF‐antagonist/angiogenesis inhibitor)‐sensitive invasion characteristic of the endometrial cancer cells. Additionally, we demonstrate a close correlation of the invasion of endometrial cancer cells with the expression and dimerization of integrin αvβ5 as well as the activation of focal adhesion kinase as the consequences of paracrine interactions. Thus, understanding of paracrine interactions of cancer cells with host stromal cells can yield new insight into the architecture and function of cancer invasion and metastasis, leading to a development of a new cancer therapeutic intervention.

Syndecan-1 overexpression promotes tumor growth and angiogenesis in an endometrial cancer xenograft model.

Objectives: Upregulation of syndecan-1, a member of the transmembranous proteoglycans that serves as a coreceptor for a wide pool of extracellular ligands, has been well documented in enabling the promotion of growth and invasion of endometrial cancer. As a step toward understanding a potential role for syndecan-1 in this process, we questioned whether syndecan-1 upregulates tumor-promoting characteristics, particularly, angiogenesis in an in vivo human xenograft tumor model. Methods: Human syndecan-1 was stably transfected into human endometrial adenocarcinoma 1A cells, and resulting transfectants were subcutaneously grafted into athymic mice; their outcomes were examined with respect to the enhancement of tumor growth and angiogenesis by immunohistochemistry, immunoblotting, and zymography. Results: Overexpression of syndecan-1 promoted tumor growth concomitant with increased angiogenesis in tumor xenografts as evidenced by an increase in immunoreactivity for vascular endothelial growth factor and vascular endothelial cell marker CD34. Furthermore, zymographic studies revealed that syndecan-1 overexpression markedly enhanced activities of matrix metalloproteinases 2 and 9. Conclusions: This is the first in vivo xenograft analysis providing evidence that supports that syndecan-1 has a critical role in carcinogenic progression, particularly, contributing to the development of angiogenesis and invasive phenotype in association with matrix metalloproteinases 2 and 9 activations in endometrial cancer.

Methyl CpG–Binding Domain Protein 3 Mediates Cancer-Selective Cytotoxicity by Histone Deacetylase Inhibitors via Differential Transcriptional Reprogramming in Lung Cancer Cells

Histone deacetylase inhibitors (HDI) have been reported to inhibit the growth and survival of cancer cells while leaving normal cells untouched. However, the mechanisms underlying this selective cell death are poorly understood. Gene expression analysis revealed that HDI treatment induced up-regulation of p21(WAF1/Cip1) and down-regulation of ErbB2 in cancer cells but not normal cells. Overexpression of p21(WAF1/Cip1) and/or silencing of ErbB2 enhanced cancer cell growth inhibition, suggesting that HDI-induced up-regulation/down-regulation of these genes play critical roles in HDI-induced growth inhibition of cancer cells. Most importantly, we found that the gene silencing factor methyl CpG-binding domain protein 3 (MBD3) was not only released from cancer-selective promoter of the HDI up-regulated p21(WAF1/Cip1) gene but also recruited to that of the HDI-down-regulated ErbB2 gene. Furthermore, silencing of MBD3 by small interfering RNA abrogated the HDI-induced gene regulation and growth inhibition in lung cancer but not in normal cells. Together, our results support the critical potential of MBD3 in HDI-induced cancer-selective cell death via cancer differential gene expression.

Comparison of in vitro maturation media of immature oocytes: the effectiveness of blastocyst culture media

OBJECTIVE To compare three different in vitro maturation (IVM) media for immature oocytes. DESIGN Experimental study. SETTING In vitro fertilization laboratory. ANIMAL(S) BDF1 female and male mice. INTERVENTION(S) Retrieval and maturation of cumulus-enclosed germinal vesicle-stage oocytes according to one of three protocols: group A, conventional IVM medium; group B, blastocyst culture medium; and group C, tissue culture medium (TCM) 199. MAIN OUTCOME MEASURE(S) Maturation, fertilization, and developmental rates of immature oocytes. RESULT(S) A total of 653 immature oocytes were cultured in vitro and then analyzed. No difference was found in maturation rates and fertilization rates in comparing groups A and B. However, the IVM rates were statistically significantly increased in groups A and B compared with group C. No difference was found in fertilization rates between media, but the developmental competency to blastocyst stage was statistically significantly higher in group B compared with group C. CONCLUSION(S) The developmental competency of immature oocytes did not differ between conventional IVM medium and blastocyst culture medium, but TCM-199 was found to be unsuitable. Evidence from mice as test subjects suggests that both conventional IVM medium and blastocyst culture medium are suitable for IVM, and that blastocyst culture medium may be a good choice for conventional IVM of immature oocytes.

Evaluation of in vitro spermatogenesis using poly(D,L-lactic-co-glycolic acid) (PLGA)-based macroporous biodegradable scaffolds

Successful in vitro differentiation of spermatogenic cells into spermatids appears to offer extremely attractive potential for the treatment of impaired spermatogenesis and male infertility. Experimental evidence indicates that biocompatible polymers may improve in vitro reconstitution and regeneration of tissues of various origins. Here, we fabricated highly porous biodegradable poly(D,L‐lactic‐co‐glycolic acid) or PLGA co‐polymer scaffolds by combining the gas‐foaming and salt‐leaching methods, using ammonium bicarbonate as a porogen, which allowed us to generate polymer scaffolds with a high density of interconnected pores of 400–500 µm in average diameter, concomitant with a high malleability to mould a wide range of temporal tissue scaffolds requiring a specific shape and geometry. The PLGA scaffolds were biocompatible and biodegradable, as evidenced by the fact that they survived almost 3 month long subcutaneous xenografting into immunodeficient host mice and became easily destroyable after recovery. Immature rat testicular cells that were seeded onto the surface of the scaffold exhibited about 65% seeding efficiency and up to 75% viability after 18 days in culture. Furthermore, our scaffolds enhanced the proliferation and differentiation of spermatogenic germ cells to a greater extent than conventional in vitro culture methods, such as monolayer or organ culture. Taken together, an implication of the present findings is that the PLGA‐based macroporous scaffold may provide a novel means by which spermatocytes could be induced to differentiate into presumptive spermatids. Copyright

Inhibitory actions of mibefradil on steroidogenesis in mouse Leydig cells: involvement of Ca(2+) entry via the T-type Ca(2+) channel.

Intracellular cAMP and Ca(2+) are involved in the regulation of steroidogenic activity in Leydig cells, which coordinate responses to luteinizing hormone (LH) and human chorionic gonadotropin (hCG). However, the identification of Ca(2+) entry implicated in Leydig cell steroidogenesis is not well defined. The objective of this study was to identify the type of Ca(2+) channel that affects Leydig cell steroidogenesis. In vitro steroidogenesis in the freshly dissociated Leydig cells of mice was induced by hCG incubation. The effects of mibefradil (a putative T-type Ca(2+) channel blocker) on steroidogenesis were assessed using reverse transcription (RT)-polymerase chain reaction analysis for the steroidogenic acute regulatory protein (StAR) mRNA expression and testosterone production using radioimmunoassay. In the presence of 1.0 mmol L(-1) extracellular Ca(2+), hCG at 1 to 100 IU noticeably elevated both StAR mRNA level and testosterone secretion (P < 0.05), and the stimulatory effects of hCG were markedly diminished by mibefradil in a dose-dependent manner (P < 0.05). Moreover, the hCG-induced increase in testosterone production was completely removed when external Ca(2+) was omitted, implying that Ca(2+) entry is needed for hCG-induced steroidogenesis. Furthermore, a patch-clamp study revealed the presence of mibefradil-sensitive Ca(2+) currents seen at a concentration range that nearly paralleled those inhibiting steroidogenesis. Collectively, our data provide evidence that hCG-stimulated steroidogenesis is mediated at least in part by Ca(2+) entry carried out by the T-type Ca(2+) channel in the Leydig cells of mice.