Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have recently shown that the integrity of the polyamine pathway is essential but not sufficient for expression of the mitogenic effect of estradiol (E2) in the N-nitrosomethylurea-induced rat mammary tumor grown in vitro in the soft agar clonogenic assay. To elucidate the mechanism of E2 action in this system, we tested whether E2 may stimulate tumor growth through induction of secretory growth factor(s), as already proposed for human breast cancer cell lines in culture. Furthermore, we investigated the potential interaction between such "autocrine" control of tumor growth by E2 and the polyamine pathway. Conditioned medium obtained from E2-treated tumors (E2-CM) but not from control tumors (C-CM) consistently stimulated colony formation when added to N-nitrosomethylurea-mammary tumors plated in soft agar under serum-free media conditions. Such growth-promoting effects of the E2-CM was found to increase with increasing protein concentrations of the medium and was abolished by pretreatment of the medium with concanavalin A, heat, and trypsin. The addition of the polyamine biosynthetic inhibitor alpha-difluoromethylornithine (1 mM) totally abolished the colony-stimulating effect of the E2-CM. Exogenous administration of spermidine (0.1 mM) reversed the inhibitory effect of alpha-difluoromethylornithine on colony formation and restored the action of the E2-CM. Although the addition of polyamines alone did not affect the number of colonies formed, the administration of spermidine was found to significantly enhance in a dose-dependent fashion the colony-stimulating effect of suboptimal concentrations of E2-CM. Attempts to identify the E2-inducible growth factor in the E2-CM and in N-nitrosomethylurea-mammary tumor specimens using monoclonal antibodies raised against the Mr 52,000 E2-inducible protein gave negative results. We conclude that autocrine stimulation of tumor growth by E2 is not limited to human breast cancer cell lines but also occurs in individual experimental mammary tumors grown in soft agar. Our results show that the polyamines must be present for the expression of this "autocrine" control of tumor growth by E2. Finally, the identity of the E2-induced growth factor operating in our system remains to be determined.
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PMID:Autocrine stimulation by estradiol-regulated growth factors of rat hormone-responsive mammary cancer: interaction with the polyamine pathway. 308 Dec 52

These experiments were designed to test whether autocrine/paracrine mechanisms are involved in the growth-promoting action of progesterone (Pg) in the N-nitrosomethylurea-induced rat mammary tumor cultured in vitro in soft agar clonogenic assay. In support of our hypothesis, we observed that conditioned media obtained from Pg-treated tumors (Pg-CM), consistently stimulated colony formation in our system to the same degree as Pg itself (approximately 140% of control). Treatment with heat, trypsin, and concanavalin A abolished the colony-stimulating effect of Pg-CM, thus suggesting the possible glycoprotein nature of the Pg-inducible growth factor(s). The growth-promoting action of Pg-CM was rather specific since CMs obtained from tumors exposed to a variety of other steroid hormones rarely stimulated colony formation and usually only to a modest degree. Administration of the polyamine biosynthetic inhibitor, alpha-difluoromethylornithine, abolished the colony-stimulating effect of Pg-CM. The inhibitory effect of alpha-difluoromethylornithine was reversed in a dose-dependent fashion by exogenous administration of spermidine, which entirely restored the growth-promoting action of Pg-CM. Addition of increasing amounts of spermidine, however, did not potentiate Pg-CM action under our experimental conditions. Our results indicate that autocrine/paracrine mechanisms may mediate, at least in part, the colony-stimulating effect of Pg in our system. The polyamine pathway plays an essential role in the expression of such control of tumor growth by Pg.
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PMID:Polyamines and autocrine control of N-nitrosomethylurea-induced rat mammary tumor growth in vitro by progesterone. 313 Jan 83

Demineralized extracts of bone matrix and conditioned media from cultured fetal rat calvaria have been reported to contain growth stimulatory activity for bone cells. To investigate the potential role of these local bone growth factors in the development of bone metastases, we chose the Walker 256 carcinosarcoma, a rat mammary tumor which causes osteolytic bone metastases and hypercalcemia. 45Ca-labeled, 19-day fetal Sprague-Dawley rat calvaria were cultured for 96 hours in BGJb medium. Walker cells from ascites tumors or cultures were grown in unconditioned media or in conditioned media harvested from the bone cultures, in the presence of 10% fetal calf serum. Media were changed every 2 days, cells were counted daily for 5 days, and 3H-thymidine uptake into acid insoluble residues was measured. The growth of tumor cells was 5-6-fold greater in conditioned media than in unconditioned media and the effect was dose dependent. Cells cultured in conditioned media demonstrated a approximately 3-fold enhancement of 3H-thymidine incorporation. Generation of growth stimulatory activity correlated with the extent of bone resorption, measured by release of 45Ca from the fetal parietal bones (r = 0.85; P less than 0.001). Conditioned media from bones cultured with 10(-7) M prostaglandin E2 (PGE2) contained greater amounts of growth stimulatory activity than untreated conditioned media, but PGE2 itself did not stimulate tumor cell growth. Addition of 3.5 mM PO4 to bone cultures blocked bone resorption and the generation of growth factors. Growth stimulatory activity was stable to heat (56 C for 30 minutes) and trypsin digestion, with an apparent molecular weight of less than 17,000 daltons by high-performance liquid chromatography. Conditioned medium also stimulated the growth of 13762 rat mammary adenocarcinoma cells, MB-MDA-231 human breast carcinoma cells, TE-85 osteosarcoma cells, a murine fibrosarcoma and rat embryonic fibroblasts, with the most potent effects noted for Walker tumor cells, the TE-85 osteosarcoma, and human breast carcinoma lines. These results suggest a mechanism by which bone resorption could promote the development of skeletal metastasis.
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PMID:Resorbing bone stimulates tumor cell growth. A role for the host microenvironment in bone metastasis. 345 36

Mammalian cells growing as multicell spheroids, an in vitro model of tumor microregions, have been shown previously to be more resistant than single cells from monolayer cultures to killing by ionizing radiation, hyperthermia, ultrasound, and chemotherapeutic drugs. Although the mechanisms by which cells in spheroids acquire these increased resistances are unknown, available evidence has indicated that intercellular contact mediates the process for ionizing radiation. This investigation was undertaken to evaluate the role of intercellular contact produced during growth of small spheroids on the sensitivity of EMT6/Ro mouse mammary tumor cells to moderate hyperthermia. Increased thermoresistance developed in small spheroids (approximately 70 micron diameter, 25 cells/spheroid), as measured by colony formation, after exposures to different temperatures in the range of 37 to 45 degrees C for periods less than or equal to 2 hr and at 42.5 degrees C for less than or equal to 8 hr. Experiments were performed to determine the relative contributions to this increased thermoresistance of 1) the extent of intercellular contact in spheroids of different cellular multiplicities, 2) differences in membrane damage influenced by trypsin heat treatment sequence, and 3) physiological changes associated with growth of cells as spheroids in suspension compared to monolayer culture. Treatment with trypsin prior to heating sensitized cells to killing by hyperthermia but did not account for the differential thermoresistance between cells from spheroids and monolayers. Spheroid multiplicity in the range of 1.16 to 76.2 cells/spheroid had no significant effect on cell survival after hyperthermia. However, cells grown in spinner suspension culture were more thermoresistant than cells from monolayer cultures and nearly as thermoresistant as cells in spheroids. From these data we conclude that the greater thermoresistance of EMT/Ro cells in spheroids is the result of cellular physiological changes associated with growth in suspension and is not mediated by intercellular contact.
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PMID:Increased thermoresistance developed during growth of small multicellular spheroids. 388 62

A growth factor has been purified to homogeneity from human mammary tumors. The human mammary tumor-derived growth factor (h.MTGF) has a molecular weight of 16,000 and an isoelectric point of 8.0 and is sensitive to heat, trypsin digestion, acid, and reduction. h.MTGF was purified to homogeneity using carboxymethylcellulose 52, heparin-Sepharose, and copper-Sepharose affinity chromatography. The stimulation of proliferation in cultured rabbit fetal chondrocytes was used as the principal bioassay. Purified h.MTGF was also mitogenic for bovine corneal endothelial cells, human fibroblasts, and a human breast cancer cell line, T-47D. It is postulated that h.MTGF may play a role in the fibrovascular changes of malignant breast tumors and promote the autonomous growth of the neoplastic cell population.
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PMID:Purification and characterization of a human mammary tumor-derived growth factor. 394 3

Evidence obtained in human breast cancer cell lines in culture suggests that estradiol stimulates the synthesis of secretory proteins which may, in turn, mediate its mitogenic effect. We questioned whether a similar mechanism could mediate the growth-promoting effect of PRL in the N-nitrosomethylurea-induced rat mammary tumor grown in soft agar, where PRL exerts a dose-dependent colony-stimulating effect. Conditioned medium obtained from PRL-treated tumors, but not from control tumors, was found to exert a significant dose-dependent colony-stimulating effect when added to N-nitrosomethylurea-induced mammary tumors plated in soft agar under serum-free medium conditions. The growth-promoting action of conditioned medium from PRL-treated tumors was abolished by pretreatment with heat, trypsin, and Concanavalin-A, suggesting the possible glycoprotein nature of the oPRL-induced growth factor(s). These results provide support for the novel hypothesis that estradiol and PRL may support the growth of hormone-responsive breast cancer through a similar mechanism.
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PMID:Autocrine stimulation by prolactin of hormone-responsive breast cancer growth in culture. 404 74

The distribution, metabolism and protein-binding of [3H] corticosterone in the rat liver was studied with respect to time and sex. The maximum recovery from the cell nuclei occurred 5 min after injection of the steroid. At this time, ten times more radioactivity was recovered from the liver cell nuclei of male animals compared to females. The recovered radioactivity in the cell nuclei was identified as mainly unmetabolised corticosterone and 5 alpha-dihydrocorticosterone. The radioactivity bound to the cytosolic glucocorticoid receptor protein also appeared to consist of unmetabolised corticosterone and 5 alpha-dihydrocorticosterone. However, 5 alpha-dihydrocorticosterone was found to have very little biological activity. The glucocorticoid receptor was found to exist in two forms with Stokes radii of 6.1 and 3.6 nm, respectively, following in vivo labelling with [3H] dexamethasone. The 6.1 nm form could be converted into the 3.6 nm form by incubation with trypsin, papain, alpha-chymotrypsin or an extract of purified rat liver lysosomes. Further incubation resulted in an even smaller form with a Stokes radius of 1.9 nm. With the help of biospecific adsorption chromatography based on the differential affinity of the activated and nonactivated complexes for DNA, the 6.1 nm form of the glucocorticoid-receptor complex was purified to near homogeneity by a rapid and simple technique. An immunoglobulin fraction from serum of a rabbit immunized with a highly purified preparation of glucocorticoid receptor contained specific antibodies to the receptor. The antibodies cross-reacted with the glucocorticoid receptor from human normal lymphocytes, chronic lymphatic leukemia cells and human hippocampus. Activated glucocorticoid receptor protein binds selectively in vitro to a cloned fragment of murine mammary tumor virus DNA. In contrast, the receptor fails to bind selectively to DNA restriction fragments from E coli plasmids pBR 322 and RSF 2124 or from bacteriophages alpha and T4.
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PMID:Glucocorticoid-binding proteins in rat liver. 695 93

Effects of trypsin treatment on insulin and concanavalin A binding to, and glucose and proline transport in, dissociated R3230AC mammary adenocarcinoma cells were examined. Reduction of binding of 125I-labelled insulin was dependent on the amount of trypsin used, the temperature and the time of the incubation period. Under conditions that reduced insulin binding by greater than 75%, transport of glucose and proline was reduced by less than 15%. Scatchard analysis of insulin binding after trypsin treatment yielded slopes similar to those from cells not exposed to trypsin, assuming either two classes of receptors or an average affinity, Ke. Dissociation of bound insulin from untreated or trypsin-treated cells was enhanced by addition of excess unlabelled ligand. Insulin added in vitro, which decreased glucose transport in untreated cells, produced a decrease in glucose transport in cells treated with trypsin for 5 min (insulin binding was decreased 35%), but not in cells treated for 45 min (insulin binding was decreased 90%). Binding of the plant lectin concanavalin A was also reduced by trypsin treatment, but to a lesser extent and with a different time-course than for insulin. Scatchard analysis of the binding of concanavalin A in untreated and trypsin-treated cells yielded comparable values for Kd. The insulinomimetic actions of concanavalin A on glucose transport were abolished after brief exposure to trypsin. Pre-treatment of cells with concanavalin A reduced insulin binding and partially protected insulin receptors from trypsin digestion, but the inability to remove all of the concanavalin A precluded its use as a method to protect insulin receptors. Thus, in this rat mammary tumor, the number, but not the affinity or functional activity, of insulin receptors can be reduced by trypsin treatment without significant effects on glucose or A system amino acid transport.
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PMID:Effects of trypsin on binding of insulin and concanavalin A and on glucose and proline transport in the R3230AC mammary adenocarcinoma. 703 58

We have shown previously (D. A. Sirbasku, 1978, Proc. Natl. Acad. Sci. U.S.A., 75:3786-3790) that an estrogen-inducible growth factor activity for rat mammary and rat pituitary tumor cells can be identified in extracts of rat uteri, although at the time of that report only a limited biochemical characterization of the activity was presented. In this report, we have evaluated the growth factor activity for lipid, steroid hormone or protein-like properties. Uterine growth factor activity was assayed by measure of the increased cell number of the MTW9/PL rat mammary tumor cell line established by this laboratory and described previously (D. A. Sirbasku, 1978, Cancer Res. 38:1154-1165). Studies showed the following characteristics of growth factor activity: destroyed by trypsin treatment; labile when heated at 80 degrees C; partially denatured by 6 M guanidine or 8 M urea treatment or 50% aqueous solutions of organic solvents; inactivated by extremes of pH or overnight treatment with mild acid; not dialyzable at neutral pH; of apparent molecular weight of 70,000 daltons by G-100 Sephadex chromatography; possessing an isoelectric point of 4.8 to 5.2; not chloroform/methanol extractable; and not in any way identified as either a lipid or a steroid hormone. The data available suggest that the uterine growth factor activity is a protein or polypeptide of apparent high molecular weight, and that the activity does not directly correspond to other known growth factors.
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PMID:Properties of a growth factor activity present in crude extracts of rat uterus. 725 89

The plasma membrane fucopeptides of tumorigenic and nontumorigenic mouse mammary epithelial cells were studied. The types of cells analyzed included (a) cell lines derived from mouse mammary carcinomas of varying etiologies (viral, hormonal, chemical carcinogen), (b) a series of clonal cells lines which were nontumorigenic at lower passage levels and tumorigenic at higher passage levels, (c) normal primary cells derived from the mammary glands of pregnant or lactating animals, and (d) primary cells from tumors produced by s.c. injection of cultured mammary tumor cells into syngeneic animals. A distinctive difference was observed in the size distribution of the trypsin-sensitive surface fucopeptides from tumorigenic and nontumorigenic mammary cells; the tumorigenic cells were relatively enriched in the larger fucopeptides. The size distribution of the trypsin-sensitive surface fucopeptides was not markedly influenced by the physiological state of the cells or by cell population density. It appears that the trypsin-sensitive surface fucopeptide size pattern may be a distinguishing characteristic between tumorigenic and nontumorigenic mouse mammary epithelial cells.
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PMID:Correlation of surface fucopeptide patterns with tumorigenicity of mouse mammary epithelial cells. 730 96


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