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M Koutsilieris

Publications and source records attributed to M Koutsilieris.

At least 37 records · Page 2Linked to original sources
PubMed1996

Human endometrial cells cultured in a type I collagen gel.

OBJECTIVE: To elaborate in vitro conditions that enable epithelial and stromal cells of human endometrium to grow within a gel of collagen. STUDY DESIGN: Primary cultures of epithelial cells derived from human endometrial biopsies were dissociated and mixed with a collagen solution, and the gel was allowed to form at physiologic pH. Control cultures were grown in plastic dishes. DNA replication was assessed by 3H-thymidine incorporation, morphology by histology and cell characterization by monoclonal antibodies to cytokeratins. RESULTS: Cells grown on plastic dishes exhibited a typical monolayer arrangement, and replication was increased 1.6-fold by the addition of stromal cell-conditioned medium (50% vol/vol). After a two- to three-week period of culture within the collagen gel in the presence of either stromal cells or stromal cell-conditioned medium, epithelial cells formed circular arrangements of cuboidal to columnar cells with open lumina resembling glands. These glandlike structures were cytokeratin positive as assessed by immunohistochemistry, thereby confirming their epithelial nature. CONCLUSION: The development of differentiated epithelial structures in a three-dimensional gel provides a promising method of studying various biochemical and cellular interactions of eutopic and ectopic endometrium.

Biopsy↗
PubMed1995

Mediation of glucocorticoid receptor function by the activation of latent transforming growth factor beta 1 in MG-63 human osteosarcoma cells.

We analyzed glucocorticoid receptor function using ligand binding assays, DNA band-shift analysis and trans-activation of the murine mammary tumor virus-thymidine kinase-chloramphenicol acetyltransferase reporter gene in transiently transfected MG-63 human osteosarcoma cells. Dexamethasone increased the distribution of MG-63 cells in the G1/G0 phase of the cell cycle, thus decreasing the rate of DNA synthesis and cell growth. Its effect on MG-63 cell growth was neutralized by RU486 and anti-transforming growth factor beta 1 (TGF beta 1) antibody. In addition, (i) dexamethasone increased the levels of active TGF beta 1 in MG-63-conditioned media without significantly altering the expression of TGF beta 1 mRNA in MG-63 cells and (ii) TGF beta 1 inhibited proliferation of MG-63 cells. Therefore, we conclude that glucocorticoid receptor function is mediated by the activation of latent-TGF beta 1 in MG-63 osteosarcoma cells.

Base Sequence↗
PubMed1995

Mediation of glucocorticoid receptor function by transforming growth factor beta I expression in human PC-3 prostate cancer cells.

We investigated the role of glucocorticoids in controlling the proliferation of androgen-independent PC-3 human prostate cancer cells via the action of transforming growth factor beta 1 (TGF beta 1). The presence of glucocorticoid receptor (GR) in PC-3 cells was detected by immunoblotting analysis using a rabbit anti-GR polyclonal antibody against the synthetic human GR peptide (hGR383-393). In PC-3 cells, GR bound radiolabeled dexamethasone with an affinity similar to wild-type GR. In addition, GR-ligand complex bound radiolabeled DNA as detected by DNA band-shift analysis on gel electrophoresis and trans-activated the mouse mammary tumor virus-thymidine kinase-chloramphenicol acetyltransferase chimeric gene in transiently transfected PC-3 cells. Dexamethasone (0.1 up to 100 nM) and TGF beta 1 (0.5 up to 50 ng/ml) inhibited PC-3 cell proliferation. TGF beta 1 and dexamethasone both increased the distribution of PC-3 cells into the G1/G0 phase of the cell cycle. Platelet-derived growth factor (PDGF) stimulated the proliferation of PC-3 cells and overcame dexamethasone's inhibition of PC-3 cell growth. Dexamethasone's inhibition (10(-7) M) of PC-3 cell growth was completely neutralized by RU 486 (10(-6)M) and partly neutralized by anti-TGF beta 1 polyclonal antibody. Furthermore, dexamethasone up modulated the expression of TGF beta 1 mRNA in PC-3 cells. Because dexamethasone's inhibition was neutralized at least in part by an anti-TGF beta 1 polyclonal antibody and dexamethasone up modulated the expression of TGF beta 1 mRNA in PC-3 cells, we conclude that GR function in human PC-3 prostate cancer cells is mediated at least in part by TGF beta 1 expression.

Cell Division↗
PubMed1995

N-terminal truncated forms of insulin-like growth factor binding protein-3 in the peritoneal fluid of women without laparoscopic evidence of endometriosis. Le groupe d'investigation en gynécologie.

OBJECTIVE: To characterize and purify peritoneal mitogens able to stimulate the proliferation of human endometrial cells in vitro. DESIGN: Peritoneal fluids (PFs) from 50 patients were collected at laparoscopy and pooled (270 mL) for purification of mitogenic activity. SETTING: University infertility clinic and endocrinology of reproduction and molecular endocrinology laboratories. PATIENTS: Fifty subjects presenting for tubal ligation, pelvic pain, mass, or infertility but otherwise having no evidence of endometriosis inflammation, infection, or tumor. INTERVENTIONS: None. MAIN OUTCOME MEASURES: Assessment of mitogenic activity by 3H-thymidine incorporation into mouse embryo fibroblasts and into primary cultures of isolated epithelial and stromal cells of human endometrium. RESULTS: The PF mitogens were purified successively on carboxymethyl-sepharose and heparin-sepharose columns followed by fractionation on cartridges of C18 silica and reverse-phase high-performance liquid chromatography (HPLC). Four distinct bands were eluted from Sep-Pak, (Mississauga, Ontario, Canada) with molecular weights of 17 to 18, 20, 25, and 29 to 30 kd. The eluted fractions of Sep-Pak exerted preferential mitogenic activity on epithelial-derived human endometrial cells at an equimolar ratio with epidermal growth factor. Microsequencing of the 17 to 18, 20, 25, and 29 to 30 kd bands showed a homologous sequence with N-terminal amino acid sequences of insulin-like growth factor binding protein-3 (IGFBP-3). CONCLUSION: These data indicate that the PF of normal women without evidence of endometriosis contains N-terminal truncated forms of IGFBP-3 that mediate an apparent preferential mitogenic action on epithelial-derived endometrial cells. Therefore, they could play a role in the ectopic growth of endometrial cells.

Amino Acid Sequence↗
PubMed1995

Purification and partial sequencing of the major mitogen for human uterine smooth muscle-like cells in leiomyoma extracts.

We purified the major mitogen for human smooth muscle-like cells in leiomyoma extracts by sequential liquid chromatography on (a) carboxymethyl-Sepharose, (b) heparin-Sepharose columns, (c) cartridges of C18 silica, and (d) linear gradient reverse-phase high performance liquid chromatography. The mitogenic activity of the leiomyoma extract throughout purification was tested by tritiated thymidine incorporation and DNA content in NIH/3T3 fibroblasts and KW human smooth muscle-like cells. Purification of the leiomyoma-derived growth factor (LDGF) for KW smooth muscle-like cells confirmed that its partial NH2-terminal amino acid (aa) sequence (1-20 aa) was identical to 113-132 aa of the human cysteine-rich protein (hCRP). A synthetic peptide which was engineered based on the purified aa sequence, stimulated the proliferation and growth of KW cells. An oligonucleotide probe constructed by the cDNA of the hcrp gene that encodes this aa sequence depicted the expression of 1.9-kb LDGF mRNA in leiomyomas and myometrium. The expression of the LDGF mRNA was three to sixfold higher in leiomyomas compared with adjacent myometrium of women harboring leiomyomas by in situ hybridization analysis. These data suggest that LDGF may participate in the pathophysiology of uterine leiomyomas.

3T3 Cells↗
PubMed1994

Three-dimensional type I collagen gel system for the study of osteoblastic metastases produced by metastatic prostate cancer.

We developed a three-dimensional type I collagen gel cell culture system that allows coculturing of human MG-63 osteoblast-like cells and various human cancer cells. Inoculation of human PC-3 metastatic prostate cancer cells into this type I collagen gel containing human MG-63 osteoblast-like cells produced an osteoblastic-like reaction that presented as an increased number of MG-63 cells and increased density of type I collagen around MG-63 cells adjacent to inoculated PC-3 cells by microscope analysis. Under identical experimental conditions, inoculation of cell-free medium, human KLE endometrial adenocarcinoma cells, and Calu-1 lung cancer cells did not produce this blastic-like reaction. In situ hybridization documented the uniform expression of insulin-like growth factor I (IGF-I) and of urokinase-type plasminogen activator (uPA) mRNA in MG-63 and PC-3 cells separately cultured in this substrata. The uniform expression of uPA was also documented by immunocytochemistry using a monoclonal and a polyclonal antihuman uPA antibody. The relative expression of uPA was higher in PC-3 cells than in MG-63, KLE, and Calu-1 cancer cells. We conclude that this novel cell culture system may become a useful model to study the pathophysiology of the osteoblastic reaction in vitro.

Adenocarcinoma↗
PubMed1994

Antiandrogens inhibit human androgen receptor-dependent gene transcription activation in the human prostate cancer cells LNCaP.

Human androgen receptor (hAR) is a ligand-dependent transcription factor that mediates androgen-induced actions on target tissues. Transfection studies in the human prostate cancer cell line LNCaP examine the ability of dihydrotestosterone (DHT), hydroxyflutamide (HO-FLU), cyproterone acetate (Cypro.A), and RU 23908-10 to stimulate or to inhibit the transcription activation of mouse mammary tumor virus-bacterial chloramphenicol acetyltransferase (MMTV-CAT). DHT stimulated transcription activation of MMTV-CAT gene in LNCaP cells in a dose-dependent manner. HO-FLU, Cypro.A, and RU 23908-10, though only partially, also stimulated the transcription activation of MMTV-CAT. Despite this, 100- to 1,000-fold molar excess of all antiandrogens inhibited the agonistic activity of 10 nM DHT in this system. Receptor binding assays confirmed that HO-FLU, Cypro.A, and RU 23908-10 competed with DHT for AR binding in LNCaP cells. Western blot analysis using AR antipeptide antibodies raised in rabbits revealed the presence of two AR protein bands in LNCaP cells, following treatment with antiandrogens. Increasing doses of HO-FLU stimulated the expression of the 114-kDa AR by 2.5-fold, but did not affect the 108-kDa AR. Increasing doses of Cypro.A and RU 23908-10 decreased the levels of both the 114-kDa and the 108-kDa AR. Although the exact nature of 108-kDa and 114-kDa AR in LNCaP cells is still unknown, these data suggest that the regulatory actions of each individual antiandrogen on AR expression in LNCaP cells may be different.

Amino Acid Sequence↗
PubMed1993

Interaction of antiandrogen-androgen receptor complexes with DNA and transcription activation.

Human androgen receptor (hAR) is a ligand-dependent transcription factor that mediates androgen-induced actions on target tissues. Transfection studies in receptor deficient monkey kidney cells CV-1 in culture examine the ability of dihydrotestosterone (DHT) and of the antiandrogens hydroxylflutamide (HO-FLU), cyproterone acetate (Cypro.A) and RU 23908-10 to stimulate or to inhibit the transcription activation of mouse mammary tumor virus-bacterial chloramphenicol acetyltransferase (MMTV-CAT). CV-1 cells cotransfected with wild type hAR (hAR1-910) and MMTV-CAT, were treated with varying concentrations of DHT. DHT stimulated transcription activation of MMTV-CAT gene in a dose-dependent fashion. Cypro.A though only partially, also stimulated the transcription activation of MMTV-CAT. In the absence of steroids, HO-FLU induced the MMTV-CAT transcription in transfectants only 4% above the basal level. RU 23908-10 revealed the least agonistic activity at concentrations between 10 nM and 1 microM. Despite this, 100- to 1000-fold molar excess of all antiandrogens inhibited the agonistic activity of 10 nM DHT in this system. Receptor binding assays confirmed that HO-FLU, Cypro.A and RU 23908-10 competed with [3H]DHT for AR binding with hAR expressed in CV-1 cells. Western blot analysis using AR antipeptide antibodies raised in rabbits revealed the presence of two AR protein bands in extracts prepared from hAR1-910 transfected CV-1 cells. Incubation of labeled synthetic palindromic androgen responsive element (ARE) with the hAR containing CV-1 cell extracts followed by u.v. cross-linking demonstrated the specificity of AR-DNA interaction. Analysis by gel mobility shift assays showed that the interaction of AR-antiandrogen complexes with labeled ARE was specific.

Androgen Antagonists↗
PubMed1993

Response to flutamide withdrawal in advanced prostate cancer in progression under combination therapy.

The effect of flutamide withdrawal was studied in 40 patients with stage D2 prostate cancer showing progression of the disease after an initial and long (average 3.9 years) period of positive response to combination therapy with flutamide associated with medical (luteinizing hormone-releasing hormone agonist) or surgical castration. Using the criteria of response of the United States National Prostatic Cancer Project, 1 complete, 3 partial and 26 stable responses were observed, while 10 patients continued to have progression after discontinuation of flutamide. The average durations of previous treatment with combination therapy were 1,794 days for the 30 responders (complete, partial and stable responses), compared to 1,726 days for the 10 nonresponders. The average duration of response after the arrest of flutamide was 440 days. Serum prostate specific antigen, which was elevated in all patients, decreased by 90% or more in 19 of the 30 responders (63%) and returned to normal in 17 (57%). The concentration of testosterone binding globulin increased after discontinuation of flutamide, thus also suggesting the suppression of an androgenic influence, while the serum levels of testosterone and dihydrotestosterone, as well as their main metabolites, did not change after withdrawal of flutamide. Alteration of local sensitivity to androgens is a probable explanation for the paradoxical positive response to the arrest of flutamide suggested in the present preliminary study.

Adenocarcinoma↗
PubMed1993

Combination therapy with flutamide and the LHRH agonist [D-Trp6, des-Gly-NH(2)10]LHRH ethylamide in stage C prostatic carcinoma.

A series of 115 previously untreated patients displaying clinical stage C prostatic carcinoma with no evidence of distant metastases received combination therapy using the antiandrogen flutamide and the LHRH agonist [D-Trp6, des-Gly-NH(2)10]LHRH ethylamide; the average follow-up was 3.9 years. Twenty-eight patients showed treatment failure with a probability of disease-free survival of 91.2% at 2 years. Twenty patients died from prostate cancer and 10 from other causes, the survival probability being 93.4% at 2 years. Local control was achieved rapidly in all patients. Urinary obstruction and hydronephrosis were corrected in all cases. When compared with data obtained after single endocrine therapy (orchiectomy or oestrogens) or radiotherapy, the treatment failure rate at 2 years was more than 3.0-fold lower after combination therapy (8.8%) than monotherapy (28.4%). The death rate 2 years after the start of combination therapy was 6.6% and was on average 22.2% (3.6-fold higher) in the studies using monotherapy (orchiectomy or oestrogens) or radiotherapy. The present data suggest that treatment of prostate cancer with combination therapy before clinical evidence of dissemination of disease permits more efficient control of local disease and a decreased rate of progression to metastatic disease.

Aged↗
PubMed1993

Optimized strategy for detection of early stage, curable prostate cancer: role of prescreening with prostate-specific antigen.

In 1,002 men aged 45-80 y, 81% of the cancers detectable by serum prostate-specific antigen (PSA), digital rectal examination (DRE), and transrectal ultrasonography (TRUS) were present in a subpopulation (19% of total) identified by serum PSA above the threshold value of 3.0 micrograms/L. This study was extended to 7,350 men using serum PSA and DRE as first approach, followed by TRUS only when 1 of these 2 tests was abnormal. Because the aim of prostate cancer detection is to find cancers at an early, potentially curable stage, it is of major interest that 71.8% of evaluable cancers were clinical stage B; 8.4% and 10.7% were stages C1 and C2, respectively; only 9.2% were stage D (metastatic) at first visit while none was at stage D at follow-up visits. This study, the first performed in an unselected, unscreened population, shows that serum PSA is the most sensitive technique to identify men at high risk of having prostate cancer and that 12% more cancers can be found at first visit by doing DRE in addition to PSA. Follow-ups can be done every second year using serum PSA alone, as 97% of the cancers detected at annual follow-up by DRE + PSA were PSA+. Cancers are discovered by the present approach at an estimated cost of $2,665 per cancer. Such cancers are potentially curable in at least 80% of cases detected at first visit and in 97% of cases at follow-up. This strategy offers the possibility to improve markedly morbidity and mortality from prostate cancer, presently the second leading cause of cancer death in North American men.

Aged↗
PubMed1993

Downstaging of localized prostate cancer by neoadjuvant therapy with flutamide and lupron: the first controlled and randomized trial.

Although the only opportunity to cure prostate cancer is treatment at an early stage, radical prostatectomy has remained relatively unpopular because 40-50% of prostate cancers estimated at diagnosis as confined to the prostate are found to be at a more advanced stage following histopathological analysis of the surgical specimen. This first prospective, randomized trial investigated the potential advantages of 3-month neoadjuvant combination therapy with flutamide and lupron before radical prostatectomy vs. prostatectomy alone in early stage prostate cancer. Cancer-positive margins were reduced from 38.5% (25 of 65) in control patients to only 13.0% (10 of 77) in men who received neoadjuvant combination therapy with the antiandrogen flutamide and the luteinizing hormone-releasing hormone (LHRH) super-agonist Lupron before radical prostatectomy (p = 0.006). Moreover, comparison of the final stage determined by histopathological examination of the surgical specimen with that estimated at diagnosis showed that a more advanced stage (upstaging) was found in 53.8% of controls, but patients who received combination therapy had an opposite effect: a more favorable stage than expected at diagnosis was found in 23.4% of cases (downstaging), a 77.2% advantage of neoadjuvant combination therapy. The concern about radical prostatectomy, underestimation of stage, is thus markedly improved by 3-month neoadjuvant therapy with flutamide and a LHRH superagonist. Cancer-negative margins are expected to be accompanied by a life expectancy not different from that of men of similar age with no prostate cancer; therefore, the present data, combined with efficient detection of early stage prostate cancer, offer the basis for dramatic improvement in the morbidity and mortality of prostate cancer.

Adenocarcinoma↗
PubMed1993

Heparin-sepharose binding growth factors in peritoneal fluid of women with endometriosis.

OBJECTIVE: To characterize and to purify the growth substances for mouse embryo fibroblasts (NIH/3T3) and endometrial-like cells of the peritoneal fluid of women with endometriosis. DESIGN: A pool of 102 mL of peritoneal fluid (PF) collected from 24 women with laparoscopic evidence of endometriosis was dialyzed and thereafter chromatographed onto carboxymethyl (CM)-sepharose, heparin-sepharose, cartridges of C18 silica, and reverse-phase high-performance liquid chromatography (reverse-phase HPLC). Mitogenic activity throughout various steps of chromatography was monitored by the increase of the (3H)-thymidine incorporation into NIH/3T3 fibroblasts and enzymatically isolated epithelial cells of rabbit endometrium. RESULTS: Peritoneal fluid of women with endometriosis contained mitogen(s) for NIH/3T3 fibroblasts that were retained on and eluted from CM-sepharose, heparin-sepharose, cartridges of C18 silica, and reverse-phase HPLC columns performed sequentially. Mitogenic material purified 2,500-fold exerted bioactivity on NIH/3T3 and the enzymatically isolated epithelial cells from rabbit endometrium. CONCLUSION: Peritoneal fluid of women with endometriosis contains heparin-binding growth factors that can be purified by sequential chromatography on CM-sepharose, heparin-sepharose, and reverse-phase HPLC columns. The presence in the PF of potent mitogens for fibroblasts and endometrium-derived epithelial cells could play an important role in the pathogenesis of endometriosis.

3T3 Cells↗
PubMed1992

Pathophysiology of uterine leiomyomas.

Uterine leiomyomas is the most common benign neoplasia in women, one of the most frequent causes of infertility in reproductive years, and the leading cause for hysterectomy. The pathophysiology of uterine leiomyomas is uncertain. Therefore, therapeutic approaches have been primarily empirical. It is now well documented that growth factors control the functional and possibly the histological integrity of several tissues. Recently the presence of growth substances in uterine tissues suggested that the role of sex steroid hormones in the pathophysiology of leiomyomas may be mediated by substances influencing the proliferation of smooth muscle cells and fibroblasts. This report summarizes the data related to the pathophysiology of leiomyomas, which indicate a possible role of growth factors in uterine leiomyomas.

Chromosome Aberrations↗
PubMed1991

Mitogenic effects of insulin and insulin-like growth factors on PA-III rat prostate adenocarcinoma cells: characterization of the receptors involved.

Four transplantable cell lines (PA-I, II, III, and IV) derived from four Lobund-Wistar (L-W) rats that manifested spontaneous prostate cancer have demonstrated metastatic capacity in visceral organs. Interestingly, PA-III cells, when deposited over the scapula or calvarium of the Lobund-Wistar rat, could produce lytic and blastic reactions on rat skeleton. Since growth factors and growth factor receptors have been implicated in bone remodeling, cancer biology, and metastatic growth of cancer cells, we have examined 1) the effects of insulin and insulin-like growth factors (IGF-I and IGF-II) on the proliferation of PA-III cells; and 2) the presence of specific receptors for these peptides. IGF-I (0.5 to 100 ng/ml), IGF-II (0.5 to 100 ng/ml), and insulin (0.5 to 10 micrograms/ml) stimulated tritiated thymidine uptake and increased the number of PA-III cells in culture. Receptor studies demonstrated the presence of specific bindings sites for IGF-I and II but not for insulin. The number and affinity of the receptor sites were: IGF-I (nb = 675 fmol/100 g protein, Kd = 0.56 nmol) and IGF-II (nb = 225 fmol/100 g protein, Kd = 0.71 nmol). Molecular characterization of IGF binding sites by polyacrylamide gel electrophoresis under denaturing conditions indicated only the presence for the type I IGF receptor. The presence of the IGF-I receptor and the absence of IGF-II and insulin receptors are discussed in relation to the capacity of PA-III cells to produce bone lesions on the L-W rat.

Adenocarcinoma↗
PubMed1991

Mitogen(s) for endometrial-like cells can be detected in human peritoneal fluid.

OBJECTIVE: To characterize the presence of mitogen(s) in the peritoneal fluid (PF). DESIGN: Aliquots of PF aspirated at laparoscopy were assessed for mitogenic activity by the rate of [3H]-thymidine incorporation into the deoxyribonucleic acid of various cell lines. SETTING: Peritoneal fluids were obtained from patients having a laparoscopy for the investigation of infertility or pelvic pain or for tubal ligation. PATIENTS: Seven women with laparoscopic evidence of endometriosis (stage I and II) and six without evidence of endometriosis. INTERVENTION: None. MAIN OUTCOME MEASURE: After 24 hours of culture in absence of serum, NIH/3T3 mouse embryo fibroblasts, (KLE) human endometrial adenocarcinoma cells, and primary cultures of rabbit endometrial cells were incubated in the presence of appropriate amounts of PF or bovine serum albumin (control) for 22 hours before adding the [3H]-thymidine for 2 hours. RESULTS: Aliquots of PF containing greater than 100 micrograms/mL total protein concentration stimulated the proliferation of all cell types. This mitogenic effect was dose-dependent and was greatest on endometrial-like cells. The mitogenic activity was fully recovered after adsorption on dextran-coated charcoal and was sensitive to tryptic digestion and to heat. It could not be retained on cartridge of C18 silica and was calculated to have a molecular weight greater than 30,000 by gel permeation chromatography. CONCLUSIONS: Peritoneal fluid from women with or without endometriosis contain growth factor(s) that are proteinaceous in nature and capable of stimulating the proliferation of endometrial-like cells and fibroblasts. These mitogens could play a role in the pathogenesis of endometriosis.

Animals↗
PubMed1990

Preferential mitogenic activity for myoblast-like cells can be extracted from uterine leiomyoma tissues.

The presence of mitogen(s) in leiomyoma extracts stimulating cells with the fibroblast, myoblast, and osteoblast phenotype was documented. Mitogenic activity in leiomyoma extracts was acid stable and sensitive to tryptic digestion. Reverse-phase high-performance liquid chromatography successfully separated mitogen(s) with preferential activity for myoblast cells from mitogens with a broad type of cell specificity and from inhibitors. This leiomyoma-derived preferential activity for myoblasts was absent in identically treated myometrial and endometrial extracts. This suggests that leiomyoma-derived substances with preferential growth factor activity for myoblast-like cells may play a role in the pathophysiologic characteristics of uterine leiomyomas.

Adult↗