leupeptins and Uterine-Neoplasms

The specific question addressed in this report is whether the resistance to steroid treatment of certain tissues or tumors which appear to contain a normal quantity of steroid-binding sites may be due to structural defects in the receptors. This question may be seen as part of the more general question of whether there are intrinsic variations in the structures of receptors for a given class of steroids in different healthy tissues, in healthy vs. malignant tissues or in different types of tumors. Our experimental approach to these questions has involved the stabilization and precise physicochemical characterization of the receptors. To date, we have studied the estrogen and progestin receptors from human breast cancers and benign and malignant gynecologic specimens and the glucocorticoid receptors from several healthy and malignant rodent tissues and from normal human lymphocytes and various types of leukemic cells. Chromatographic and ultracentrifugal analyses in buffers of low ionic strength, containing 20 mM Na2MoO4 as the stabilizer, have revealed each of these receptors to be a large, oligomeric complex, characterized by remarkably similar values of the Stokes radius, sedimentation coefficient, molecular weight and axial ratio. In the absence of adequate stabilization, however, we found that the receptors for three classes of steroids in extracts of some healthy, steroid-responsive tissues, such as rat kidney and human uterine endometrium, are invariably degraded by endogenous proteinases. The extent of such cleavage is increased considerably by freezing the tissues prior to homogenization. Studies designed to distinguish the intact receptors from the products of proteolysis have included the characterization of receptors in cytosols prepared from mixtures of rat liver and kidney. The results strongly support the interpretation that the smaller size of the receptors detected in kidney cytosol reflects their cleavage by the more active proteinases in that tissue. The sizes and shapes of the receptors in cytosols from various tissues were found to be correlated with the activities of specific endopeptidases, assayed fluorometrically with peptidyl derivatives of 7-amino-4-methylcoumarin (AMC). These studies suggested that the receptors are vulnerable to cleavage by "lysine-specific" endopeptidases, detected with t-butyloxycarbonyl-L-valyl-L-leucyl-L-lysyl-AMC. An enzyme of this specificity was partially purified from rat kidney cytosol and tested for i

Topics: Animals; Breast; Breast Neoplasms; Centrifugation, Density Gradient; Cytosol; Drug Resistance; Endometrium; Endopeptidases; Female; Humans; Kidney; Leukemia; Leupeptins; Liver; Lysine; Macromolecular Substances; Male; Molybdenum; Osmolar Concentration; Protein Conformation; Rats; Rats, Inbred Strains; Receptors, Cell Surface; Receptors, Estrogen; Receptors, Glucocorticoid; Receptors, Progesterone; Substrate Specificity; Ultracentrifugation; Uterine Neoplasms

Embryo implantation involves invasion of placental extravillous trophoblast cell (EVTs) into the uterus. Hyperactive EVT invasion occurs in hydatidiform moles and choriocarcinomas. We have previously demonstrated that the 20S proteasome is involved in mouse embryo implantation and its action is mediated via regulating the expression and activities of matrix metalloproteinase (MMP)-2 and MMP-9 in the EVTs. Our objective was to investigate whether low molecular mass polypeptide-2 (LMP2), a beta subunit of the 20S proteasome, is involved in the regulation of human trophoblast invasion. Normal human placentas or placentas from hydatidiform mole patients were collected and the expression of LMP2 in different cell types including trophoblastic column (TC), cytotrophoblast cells (CTB) and syncytiotrophoblast (STB) under different pathological states were studied by immunohistochemical analysis. Furthermore, the effect of LMP2 or proteasome on cell invasion was measured by using RNAi and inhibitors in a Matrigel invasion assay system in HTR-8/SVneo cells, a human invasive extravillous trophoblast cell line. Changes in the invasion-related molecules including MMP-2 and MMP-9 were also examined by using real time PCR and gelatin zymography. We demonstrated that the expression of LMP2 in TC of partial hydatidiform mole and complete hydatidiform mole, is higher than that in TC of normal human placentas. Besides, LMP2 knockdown significantly attenuated IL-1beta-induced cell invasion in vitro, a response readily induced by proteasome inhibitors. In summary, over-expression of the 20S proteasome beta-subunit LMP2 in trophoblast cells of hydatidiform moles may contribute to its highly invasive phenotype.

Topics: Acetylcysteine; Cell Line; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Embryo Implantation; Female; Humans; Hydatidiform Mole; Immunohistochemistry; In Vitro Techniques; Interleukin-1beta; Leupeptins; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Neoplasm Invasiveness; Placentation; Pregnancy; RNA Interference; Trophoblasts; Uterine Neoplasms

Estrogen receptor alpha (ERalpha)-positive breast cancer cell lines are up to 10 times more sensitive than ERalpha-negative cell lines to the antiproliferative activity of the histone deacetylase inhibitor trichostatin A (TSA). The purpose of the study was to investigate the mechanisms underlying this differential response.. In the ERalpha-positive MCF-7 cell line, TSA repressed ERalpha and cyclin D1 transcription and induced ubiquitin dependent proteasomal degradation of cyclin D1, leading primarily to G(1)-S-phase cell cycle arrest. By contrast, cyclin D1 degradation was enhanced but its transcription unaffected by TSA in the ERalpha-negative MDA-MB-231 cell line, which arrested in G(2)-M phase. Cyclin D1 degradation involved Skp2/p45, a regulatory component of the Skp1/Cullin/F-box complex; silencing SKP2 gene expression by RNA interference stabilized cyclin D1 and abrogated the cyclin D1 down-regulation response to TSA.. Tamoxifen has been shown to inhibit ERalpha-mediated cyclin D1 transcription, and acquired resistance to tamoxifen is associated with a shift to ERalpha-independent cyclin D1 up-regulation. Taken together, our data show that TSA effectively induces cyclin D1 down-regulation through both ERalpha-dependent and ERalpha-independent mechanisms, providing an important new strategy for combating resistance to antiestrogens.

Topics: Antineoplastic Agents, Hormonal; Breast Neoplasms; Cell Cycle; Cell Proliferation; Cyclin D1; Cysteine Proteinase Inhibitors; Drug Resistance, Neoplasm; Endopeptidases; Estrogen Receptor alpha; Female; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Leupeptins; RNA Interference; S-Phase Kinase-Associated Proteins; Tamoxifen; Transcription, Genetic; Tumor Cells, Cultured; Uterine Neoplasms

In preparation for studies of progestin receptor dynamics in human uterine tissues, we have investigated the effect of tissue handling and the protease inhibitor leupeptin on the molecular forms of progestin receptors in human endometrium, myometrium, and leiomyoma. Tritiated R5020 [17,21-dimethyl-19-nor-4,9-pregnadiene-3.20-dione) (promegestone)] was the labeled ligand, and sucrose density gradient ultracentrifugation was used to determine the sedimentation coefficients of the receptors. Scatchard plots of saturation analyses were used to determine the number of binding sites and dissociation constants. We found that rapid chilling of tissue at surgery was necessary to maintain specific progestin binding in myometrium, that leupeptin permitted demonstration of 8S receptors in myometrium and leiomyoma, and that 8S receptors were present in endometrium, with or without leupeptin. With careful handling of tissue and minimal homogenization, leupeptin may not be necessary to preserve 8S receptors in myometrium and leiomyoma cytosols; however, the use of leupeptin gives greater assurance that 8S receptors will be preserved. The number of binding sites varied from 0.6-2.0 pmol/mg cytosol protein, and the Kd values observed were between 1.7-6.9 X 10(-10) M.

Topics: Centrifugation, Density Gradient; Drug Stability; Endometrium; Female; Humans; Leiomyoma; Leupeptins; Myometrium; Oligopeptides; Promegestone; Receptors, Progesterone; Specimen Handling; Uterine Neoplasms; Uterus

Topics: Cell Line; Cholesterol; Chromatography, Gel; Female; Humans; Iodine Radioisotopes; Leupeptins; Lipoproteins, HDL; Ovarian Neoplasms; Trypsin Inhibitors; Uterine Neoplasms; Vaginal Neoplasms