Biomedical subjects

M Koutsilieris

Publications and source records attributed to M Koutsilieris.

At least 73 records · Page 4Linked to original sources

PubMedDate not supplied

Leiomyoma-derived growth factors for smooth muscle cells.

The presence of growth substance(s) for smooth muscle cells and fibroblasts was documented in extracts of human leiomyoma tissues (Koutsilieris et al, Am. J. Obst. Gynecol. 163: 1665-1670, 1990). In this study leiomyoma tissues (700 grs) obtained at surgery were extracted and purified using CM-sepharose, heparin-sepharose and reverse-phase high performance liquid chromatography (r-HPLC). The mouse NIH/3T3 cell fibroblasts (ATCC-CRL 1658) and the rat smooth muscle cells A10 (ATCC-CRL 1476) were used as indicator bioassay systems assessing the proliferative effect (tritiated-thymidine incorporation and cell number) of leiomyoma extracts. The profile of peptides purified by cm-sepharose, heparin-sepharose and by r-HPLC was analyzed by standard SDS gel electrophoresis. An overall 50,000 fold purification was achieved of a preferentially acting material on rat aorta smooth muscle cells (A10 cells). This material possessed mitogenic activity equivalent to epidermal growth factor (EGF) on A10 smooth muscle cells, in vitro. We believe that the final chemical definition of such leiomyoma-derived growth substance with preferential action on smooth muscle cells may elucidate the paracrine and/or autocrine interactions among myometrial cells and thus could provide new clues for the pathogenesis of uterine leiomyomas.

3T3 Cells↗

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PA-III rat prostate adenocarcinoma cells (review).

PA-III is one of the four transplantable cell lines propagated in vitro in cell monolayer cultures from four spontaneously developed prostate cancer tumors in aged germfree and conventional Lobund-Wistar (L-W) rats (Pollard Model). Our data documented the presence of IGF-I and glucocorticoid but the absence of IGF-II, insulin and androgen receptors in PA-III cells. Activation of IGF-I receptors by IGF-I, IGF-II and insulin stimulated the proliferation of PA-III cells. Activation of glucocorticoid receptors by dexamethasone inhibited the proliferation of PA-III cells. PA-III cells contained urokinase-like activity and expressed the mRNA for urokinase gene which was negatively regulated by glucocorticoids. The above results suggest that PA-III cells could be a useful model for the study of glucocorticoid effects on prostate cancer cells. In addition, these data are discussed in relationship to the capacity of PA-III cells to produce the osteoblastic reaction when transplanted on L-W rat skeleton. A model outlining the putative paracrine interactions between PA-III cells and rat osteoblasts is proposed.

Adenocarcinoma↗

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Proteinolytic activity against IGF-binding proteins involved in the paracrine interactions between prostate adenocarcinoma cells and osteoblasts.

PA-III rat prostate adenocarcinoma cells are capable of inducing osteoblastic reaction after inoculation onto rat skeleton. In this study PA-III cells and osteoblast-derived rat osteosarcoma cells (UMR 106 cells) were employed to characterize the cellular interactions in the PA-III cell-induced bone tumors, in vitro. Insulin-like growth factor-I (IGF-I) and conditioned media (CM) of UMR 106 cells stimlated tritiated-thymidine incorporation into the DNA of PA-III cells growing in serum-free media. This effect was inhibited by monoclonal anti-hIGF-I antibody. In addition PA-III cell CM contained proteinolytic activity for the IGF-binding proteins of UMR 106 cell CM (IGFBP-1 and -2). This proteinase activity hydrolyzed also benzyloxycarbonyl-lysine thiobenzyl ester (BLT) and its action on IGFBPs and BLT was inhibited by benzamidine and aprotinin. Proteinase activity of PA-III cell CM when bound covalently to tritiated-dilsopropylfluoro-phosphate (DFP) and then analyzed on SDS-PAGE gel electrophoresis, revealed the presence of radioactivity linked with a 35 kDa protein band. This proteinase was eluted in the void volume of the G-50 sephadex column and was retained on and eluted from p-benzamidine affinity column. The 35 kDa proteinase was retained on and was eluted from cartridges of the C18 silica by 80% acetonitrile over 0.1% trifuroacetic acid. This partially purified material hydrolyzed BLT substrate and IGFBPs of UMR 106 cell CM and its effect was inhibited by benzamidine and aprotinin. These data indicate that PA-III cell CM contains a 35 kDa proteinase capable of digesting the IGFBPs and thus increases the bioavailability of osteoblast-derived IGFs. This mechanism may participate in the pathophysiology of the PA-III cell-induced bone tumor and its subsequent osteoblastic reaction.

Adenocarcinoma↗

PubMedDate not supplied

Skeletal growth factor involvement in the regulation of fracture healing process.

Skeletal growth factors are peptides that serve as signalling agents for living cells, thereby participating in the autocrine, paracrine, intracrine and endocrine bioregulation of tissues and organs in human physiology. Growth factors elicit their cellular actions after binding to specific receptors, which are large transmembrane proteins located on target cells. These receptors relay signals via specific intracellular signal transduction pathways capable of regulating gene transcription, thereby modifying cell proliferation, cell function, cell differentiation and apoptosis. Notably, growth factors and their specific receptors are expressed in and around a bone fracture repair site, suggesting strongly that they play a significant role in the physio/pathology of fracture healing. Conceivably, fine adjustments of specific growth factor activity during the different stages of the fracture healing process can serve as potential therapeutic targets, enhancing bone repair capacity and reducing irregularities of the healing process.

Animals↗

PubMedDate not supplied

Epoetin beta (NeoRecormon) corrects anaemia in patients with hormone-refractory prostate cancer and bone metastases.

BACKGROUND: Severe anaemia is common in patients with metastatic, hormone-refractory prostate cancer (HRPC). PATIENTS AND METHODS: We evaluated the efficacy of epoetin beta in correcting anaemia and maintaining haemoglobin (Hb) levels in this group of patients. Patients with HRPC, bone metastases and anaemia (Hb < 12 g/dl) were included. Epoetin beta, 30,000 IU per week in three divided doses, was administered subcutaneously, with iron supplementation when needed. If Hb increased by < 1 g/dl during the first 4 weeks of therapy the epoetin dose was increased (increments of 5,000 IU per dose) at fortnightly intervals to a maximum of 60,000 IU per week. Patients with haematopoietic response (Hb increase > or = 2 g/dl from baseline or Hb level > or = 12 g/dl without blood transfusions) went on to receive epoetin beta 10,000 IU once weekly for up to 24 weeks. RESULTS: All 29 evaluable patients demonstrated a haematopoietic response to epoetin beta treatment. None of the patients required blood transfusions. All patients showed improvements in quality of life (assessed using the EORTC QLQ-C30 questionnaire). Hb levels were maintained for the remainder of the trial. Epoetin beta was very well tolerated. CONCLUSION: Epoetin beta therapy resulted in a rapid and sustained improvement in Hb levels in patients with HRPC metastatic to bone.

Aged↗

PubMedDate not supplied

Three-dimensional type I collagen cell culture systems for the study of bone pathophysiology.

Three-dimensional (3-D) type I collagen cell culture systems composed of reconstituted collagen fibres are able to support short- and long-term growth of various cell types, including cancer cell lines, endothelial cells, endometrial cells, hepatocytes, osteoblasts and fibroblasts and to sustain or even enhance cell differentiation, in vitro. In addition, 3-D culture systems have been successfully used in the investigation of complex biological processes, such as angiogenesis, wound healing, tumour invasion and metastasis. The latter suggested that 3-D culture systems have the potential to simulate cell-cell interactions, which take place in tissues under physiological and pathophysiological conditions. This review focuses on the investigational use of 3-D collagen cell culture systems in bone physiology and the pathophysiology of skeletal metastasis.

Animals↗

PubMedDate not supplied

Carotid body paraganglioma and SDHD mutation in a Greek family.

BACKGROUND: Carotid body (CB) is a highly specialized paraganglion originating from the neural crest ectoderm. CB paraganglion can be caused either by a genetic predisposition (hereditary paraganglia) or by chronic hypoxic stimulation. Germline mutations in any of the following genes: SDHD, SDHC, SDHB, PGL2 or other unknown genes, can cause paragangliomas (PGLs). MATERIALS AND METHODS: We studied a Greek family in which the two daughters had carotid body paraganglioma, whereas both parents did not. RNA extraction, reverse transcriptase polymerase chain reaction and direct DNA sequencing were performed, in order to identify SDHD mutations in all four exons. RESULTS: Our results revealed the existence of the missense mutation Y114C, in exon-4 of the SDHD gene, in the unaffected father and both affected sisters. CONCLUSION: DNA testing was performed, for the first time in Greece, on patients with carotid body tumor. This marks a new geographical location, in the literature, for this mutation.

Adult↗

PubMedDate not supplied

The expression of mRNA for glucocorticoid receptor gene and functional glucocorticoid receptors detected in PA-III rat prostate adenocarcinoma cells.

The metastatic capacity of PA-III rat prostate adenocarcinoma cells has been well documented, although little is known about the biological and biochemical characteristics of PA-III cells. This study characterizes PA-III cells with regard to the presence or absence of glucocorticoid and androgen receptors. Cytosols of PA-III cells possessed [3H]-dexamethasone binding sites with association constant (Ka) 0.46 +/- 0.17 x 10(9) M-1 and number 341 +/- 175 fmols/mg protein. Displacement of [3H]-dexamethasone binding from PA-III cytosols achieved by increasing doses of unlabelled dexamethasone, corticosterone, cortisol, progesterone, deoxycorticosterone and aldosterone documented glucocorticoid binding specificity. Northern and dot blot analyses detected the expression of mRNA for glucocorticoid receptor using a 750 bp cDNA probe of the glucocorticoid receptor gene. Twenty-four hours incubation of PA-III cells with increasing amounts of dexamethasone resulted in a remarkable inhibition of the growth of PA-III cells. Binding studies with [3H]-R1881 as well as dot blot and Northern blot analyses using a 500 bp cDNA probe of androgen receptor gene could not detect the presence of androgen receptors in PA-III cells. The present study documented functional glucocorticoid receptors in the androgen-independent PA-III rat prostate adenocarcinoma cells. These results suggest that glucocorticoids may regulate important aspects of androgen-independent prostate cancer cells growth and functions.

Adenocarcinoma↗

PubMedDate not supplied

Characterization and partial purification of the 8-9S androgen receptor from benign prostatic hyperplasia tissues.

The present study reports a 3,800-fold purification of the 8-9S androgen-receptor complex from benign prostate hyperplasia (BPH) tissues using differential chromatography. In addition, the BPH androgen receptor complexes have been characterized using sucrose density gradient (SDG) ultracentrifugation, gel permeation, and anion exchange high performance liquid chromatography (HPLC). Results indicate that a) under nontransforming conditions, BPH cytosols contained both 8-9S (40-78%) and 4S (22-60%) androgen-receptor forms, b) apparent molecular weights of these androgen-receptor apparent molecular weights of these androgen-receptor complexes, as analyzed by gel permeation HPLC, were estimated to correspond at 270 kDa, and 90 kDa respectively, c) 8-9S androgen-receptor complexes were retained on an anion exchange HPLC column and could be eluted at 0.22 M KCl at a linear gradient, whereas 4S complexes were not retained on anion exchange columns under identical experimental conditions, d) 10X dilution of BPH cytosols containing only the 4S (0.6 M KCl) form and subsequent chromatography on anion exchange HPLC system was indicative of fragmentation (these fragments were retained on anion exchange columns and could be eluted by 0.33 M KCl on a linear gradient HPLC), and e) increased temperature (22 C) was permissive of proteolytic fragmentation (fragments were estimated to correspond at 30, 15, and 5 kDa). The results are discussed in relationship with the composition of the nontransformed androgen-receptor molecules.

Aged↗

PubMedDate not supplied

Uterus-derived growth substances: possible implications for human uterus physiology and pathophysiology (review). The Group for Gynecological Investigation.

The presence of mitogens and inhibitors in myometrial, endometrial and leiomyoma extracts was documented. Growth substances of human uterus extracts were sensitive to tryptic digestion and were acid stable. Mitogens and inhibitors were retained and could be eluted from cartridges of octadecylsilyl-silica by 80% acetonitrile. Reverse-phase high performance liquid chromatography (r-HPLC) could separate mitogens from inhibitors. In addition, r-HPLC profiles revealed eluates of myometrial and leiomyoma extracts containing mitogens with preferential activity for either osteoblasts or myoblast-like cells, respectively. The presence of mitogens and inhibitors in human uterine extracts suggests a possible involvement of these growth substances in the regulation of various physiological and pathophysiological processes of human uterus.

Chromatography, High Pressure Liquid↗

PubMedDate not supplied

Characterization of human uterus-derived growth substances.

Human myometrium and endometrium extracts processed separately could stimulate tritiated-thymidine incorporation into the DNA of cells with the fibroblast, myoblast and osteoblast phenotype. This effect was dose-dependent. Mitogenic activity of myometrial and endometrial extracts was acid stable and sensitive to tryptic digestion. In addition, mitogens of myometrial and of endometrial extracts were retained and could be eluted by 80% acetonitrile over 0.1% trifluoroacetic acid from cartridges of octadecylsilyl-silica. Reverse-phase high performance liquid chromatography (r-HPLC) separated fractions of myometrial extracts with broad cell and preferential cell type specificity from fractions with inhibitory activity for detector cells. Reverse-phase HPLC revealed the presence in endometrial extracts of various fractions with mitogenic activity for all detector cells. These growth and inhibitory factors could mediate important autocrine, paracrine and/or endocrine regulatory processes of human uterus.

Animals↗

PubMedDate not supplied

The assessment of disease aggressivity in stage D2 prostate cancer patients (review).

Suppression of androgen levels in blood of stage D2 prostate cancer patients has been the prominent treatment for advanced prostate cancer. However, the duration of hormone sensitivity of prostate tumor is variable. The type of initial response to hormonal treatment, the length of response and patient's survival are in direct association with disease aggressiveness. Recently, an arithmetic formula expressing disease aggressivity was computed using pretreatment values of prostatic acid phosphatase (P.A.P.), alkaline phosphatase (A.P.), degree of tumor differentiation and number of bone metastases. This aggressiveness score was related to disease response and patients outcome receiving hormonal treatments. The use of an arithmetic formula to express disease aggressivity could result in a subdivision of the disease. The identification of the subgroup of stage D2 patients destined not to benefit from hormonal manipulation could change the strategies employed up until today for the treatment of advanced prostate cancer.

Estrogens↗

PubMedDate not supplied

Sucrose density gradient and exchange high performance liquid chromatography characteristics of rat prostate androgen receptor forms.

In this study, we characterized rat prostate androgen receptor using sucrose density gradient ultracentrifugation, gel permeation and anion exchange high performance liquid chromatography (HPLC). Our results indicated that 1) androgen receptor complexes in rat prostate cytosols containing no KCl were of 8-9S form corresponding to 270kDa, and in cytosols containing 0.6M KCl were of 5-6S form corresponding to 90kDa; 2) 8-9S androgen receptor complexes were retained and could be eluted by 0.22M KCl in a linear gradient anion exchange HPLC; 3) Dilution (10X) of the 8-9S and of the 5-6S receptor complexes and subsequent chromatography on anion exchange columns was indicative of fragmentation. Fragments were of 3-4S form and were eluted by 0.33M KCl on anion exchange HPLC. Temperature (22 degrees C) was permissive of proteolysis generating a 30kDa androgen receptor fragment. These results are discussed in the context of the composition of non-transformed rat prostate androgen receptor.

Animals↗

PubMedDate not supplied

Characterization of androgen receptor by high performance liquid chromatography and sucrose density gradient ultracentrifugation in normal and malignant human prostatic tissues.

In the present study we have characterized androgen-receptor complexes of normal and malignant human prostate cytosols using sucrose density gradient centrifugation, gel permeation and anion exchange high performance liquid chromatography (HPLC). Our results indicated that: 1) malignant tissue cytosols differed from normal by the presence of a 4-5S androgen receptor form which accounted for 30% of total specific-binding of malignant tissue cytosols, 2) 8-9S androgen-receptor complexes in normal and malignant prostate cytosols were estimated as 270kDa by gel permeation HPLC, 3) 8-9S complexes were retained and could be eluted by 0.22M KCl on a linear gradient anion exchange HPLC, 4) 4-5S androgen-receptor complexes were estimated as 90kDa by gel permeation HPLC and were not retained on anion exchange HPLC in our experimental conditions, and 5) either 10X dilution of the 4-5S complexes and subsequent anion exchange HPLC, or anion exchange chromatography of 8-9S complexes at 22 degrees C were causing fragmentation of the androgen receptor molecule from normal and malignant tissues. These fragments had enhanced affinity for anion exchange columns. These results are discussed in relation to the composition of the nontransformed androgen receptor macromolecule.

Adult↗

PubMedDate not supplied

Prostate-derived growth factors for bone cells: implications for bone physiology and pathophysiology: review.

Metastatic prostatic cancer is virtually unique in its tendency to induce osteoblastic lesions. Several theories have been proposed for the explanation of this phenomenon, among them, the hypothesis of growth substances secreted locally by the metastatic prostatic cancer cells. Thus the present review summarizes today's stage of knowledge on bone cell physiology and presents the results of several studies which have tested the mitogenic capacity of extracts from prostatic cancer tissues on various isolated cell culture systems.

Adenocarcinoma↗

PubMedDate not supplied

Urokinase-type plasminogen activator: a paracrine factor regulating the bioavailability of IGFs in PA-III cell-induced osteoblastic metastases.

The transplantation of PA-III rat prostate cancer cells onto rat skeleton produces osteoblastic metastases. Therefore w e studied the paracrine interactions between the PA-III cells and osteoblast-derived osteosarcoma cells (UMR 106 cells). A serine protease secreted by PA-III cells hydrolyzed IGF-binding protein-1 and IGF-binding protein-2 (IGFBP-1 and IGFBP-2) detected in the cell culture media (CM) of OMR 106 cells by western ligand blotting. The serine protease of PA-III cell CM was purified using a benzamidine affinity column. This protease was a protein of 45-50 kDa on polyacrylamide gel electrophoresis under non-reducing conditions but generated two protein bands under reducing conditions; a) one of 33-35 kDa possessing protease activity and b) another of 20-25 kDa which was proteinolytically inactive. Sequence analysis identified the amino acid sequence of the a-chain (20-25 kDa band) and of the b-chain (33-35 kDa band) of rat urokinase-type plasminogen activator molecule. Urokinase purified from PA-III cell CM hydrolyzed IGFBPs of UMR 106 cells and stimulated the proliferation of UMR 106 cells in serum-free cultures. Its protease activity was abolished by benzamidine and aprotinin. Its mitogenic activity for osteoblasts was inhibited by anti-IGF-I monoclonal antibody. Northern blot analysis documented the expression of the urokinase-type plasminogen activator gene in the mRNA extracted from PA-III cells. Urokinase expression was inhibited by dexamethasone. Therefore, we conclude that urokinase-type plasminogen activator stimulates osteoblasts via an IGF-I dependent mechanism. Hydrolysis of the IGFBOPs at the sites of PA-III cell-induced bone tumors account for an increased bioavailability of IGFs. This may facilitate the development and the growth of PA-III cell-induced bone tumor and can also mediate the subsequent local osteoblastic reaction.

Animals↗

PubMedDate not supplied

Characterization of KW smooth muscle-like human myometrial cells.

We characterized the human KW cell line to investigate whether it can serve as a model to study uterine muscle physiology in vitro. KW cells stained (a) positive for vimentin, smooth-muscle-specific alpha actin, tissue-type plasminogen activator (tPA), urokinase-type PA (uPA), uPA receptor, PA inhibitor 1, latent transforming growth factor beta 1 (latent TGF-beta 1) and 17 beta-hydroxysteroid dehydrogenase type I, and (b) negative for desmin, endoglin and cytokeratin 19. Insulin-like growth factor I, epidermal growth factor and platelet-derived growth factor stimulated the DNA synthesis in KW cells in a dose-dependent manner. Therefore, KW cells express a phenotype compatible with human uterine muscle cells. Hence, they can serve as a model to study uterine muscle physiology in vitro.

17-Hydroxysteroid Dehydrogenases↗

PubMedDate not supplied

Stratification of stage D2 prostate cancer patients by a disease aggressiveness score and its use in evaluating disease response and outcome to combination hormonal treatment (GnRH-A plus flutamide).

Suppression of androgen levels of stage D2 prostate cancer patients has been the prominent treatment for advanced prostate cancer. An arithmetic formula expressing disease aggressivity computed by the pre-treatment levels of alkaline phosphatase (AP), degree of tumor differentiation and number of bone metastases correlated well with disease response and outcome in stage D2 patients treated by chronic administration of the gonadotropin-releasing hormone agonistic analogues (GnRH-As; buserelin) or orchiectomy. In the present study we analyzed retrospectively disease response and outcome in 262 previously untreated stage D2 prostate cancer patients who received combination hormonal treatment (GnRH-A plus flutamide) using this aggressiveness score. Mean value of this aggressiveness score between patients who were grouped with reference to type of disease response according to the criteria established by the National Prostatic Cancer Project (NPCP) was statistically different (Kruskal-Wallis, p > 0.001). Pairwise comparison documented that this was true between all but the stable and progression groups. Linear regression analysis documented significant correlation of this aggressiveness score with length of response and survival (r = 0.59, and 0.45, respectively: p > 0.0001). Analysis of slopes obtained by linear regressions between aggressiveness score and survival in patients treated with GnRH-A plus flutamide and with GnRH-A monotherapy documented that disease response and outcome was superior among patients who received combination hormonal treatment. These data indicate that this aggressiveness score correlated well type of disease response, length of response, and survival with patients given combination hormonal treatment, and as such it can be used as an objective tool for analyzing clinical data.

Analysis of Variance↗