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N Olea

Publications and source records attributed to N Olea.

At least 55 records · Page 3Linked to original sources
PubMed1995

The E-screen assay: a comparison of different MCF7 cell stocks.

MCF7 human breast cancer cells have been studied extensively as a model for hormonal effects on breast cancer cell growth and specific protein synthesis. Because the proliferative effect of natural estrogen is considered the hallmark of estrogen action, it was proposed that this property be used to determine whether a substance is an estrogen. The E-screen assay, developed for this purpose, is based on the ability of MCF7 cells to proliferate in the presence of estrogens. The aim of our study was to characterize the response of four MCF7 cell stocks (BUS, ATCC, BB, and BB104) and determine which of them performed best in the E-screen test. The four stocks assayed were distinguishable by their biological behavior. In the absence of estrogen, MCF7 BUS cells stopped proliferating and accumulated in the G0/G1 phase of the cell cycle; estrogen receptors increased, progesterone receptors decreased, and small amounts of pS2 protein were secreted. Of all the MCF7 stocks tested, MCF7 BUS cells showed the highest proliferative response to estradiol-17 beta: cell yields increased up to sixfold over those of nontreated cells in a 144-hr period. The differences between estrogen-supplemented and nonsupplemented MCF7 BUS cells were due mostly to G0/G1 proliferative arrest mediated by charcoal dextran-stripped serum. MCF7 BUS cell stocks and others showing a similar proliferative pattern should be chosen for use in the E-screen test, or whenever a proliferative effect of estrogen is to be demonstrated.

Benzhydryl Compounds↗
PubMed1995

The E-SCREEN assay as a tool to identify estrogens: an update on estrogenic environmental pollutants.

Estrogens are defined by their ability to induce the proliferation of cells of the female genital tract. The wide chemical diversity of estrogenic compounds precludes an accurate prediction of estrogenic activity on the basis of chemical structure. Rodent bioassays are not suited for the large-scale screening of chemicals before their release into the environment because of their cost, complexity, and ethical concerns. The E-SCREEN assay was developed to assess the estrogenicity of environmental chemicals using the proliferative effect of estrogens on their target cells as an end point. This quantitative assay compares the cell number achieved by similar inocula of MCF-7 cells in the absence of estrogens (negative control) and in the presence of 17 beta-estradiol (positive control) and a range of concentrations of chemicals suspected to be estrogenic. Among the compounds tested, several "new" estrogens were found; alkylphenols, phthalates, some PCB congeners and hydroxylated PCBs, and the insecticides dieldrin, endosulfan, and toxaphene were estrogenic by the E-SCREEN assay. In addition, these compounds competed with estradiol for binding to the estrogen receptor and increased the levels of progesterone receptor and pS2 in MCF-7 cells, as expected from estrogen mimics. Recombinant human growth factors (bFGF, EGF, IGF-1) and insulin did not increase in cell yields. The aims of the work summarized in this paper were a) to validate the E-SCREEN assay; b) to screen a variety of chemicals present in the environment to identify those that may be causing reproductive effects in wildlife and humans; c) to assess whether environmental estrogens may act cumulatively; and finally d) to discuss the reliability of this and other assays to screen chemicals for their estrogenicity before they are released into the environment.

Animals↗
PubMed1995

Interaction between ionizing radiation, estrogens and antiestrogens in the modification of tumor microenvironment in estrogen dependent multicellular spheroids.

MCF7 human breast cancer cells growing as multicellular spheroids were examined as a model of three-dimensional cellular organization. Estrogen-free medium inhibited spheroid formation. In medium containing estrogens, the antiestrogen hydroxytamoxifen decreased the spheroid growth rate. Analyses with the recursion formula after Gompertz fitting showed that the rate of exponential decrease in growth rate (alpha) was alpha 0.099 +/- 0.013 d-1, and the decrease in alpha' was 0.061 +/- 0.015 d-1 for 0.1 microM hydroxytamoxifen and control spheroids respectively. MCF7 cells which had been growth arrested in an estrogen-free medium showed a significant decrease in radiosensitivity (surviving fraction at 2 Gy, SF2 = 63%) when compared with 0.1 nM 17 beta-estradiol-treated cells (SF2 = 38%). No differences in radiosensitivity were seen in MCF7 spheroids in estrogen-supplemented medium (radiation dose necessary to control 50% of spheroids (SCD50) was 5.51 Gy; derived alpha, beta and SF2 were 0.301 +/- 0.110 Gy-1, 0.018 +/- 0.005 Gy-2, and 51% respectively) when compared with monolayer cultures in the same medium (alpha = 0.316 +/- 0.059 Gy-1, beta = 0.023 +/- 0.006 Gy-2 and SF2 = 50%). In the spheroid model, manipulating the cellular environment, i.e., with estrogen treatment, modulates sensitivity to ionizing radiation.

Breast Neoplasms↗
PubMed1995

Development of a marker of estrogenic exposure in human serum.

The deleterious, disruptive effects of estrogen mimics on the endocrine system were discovered after the compounds were released into the environment. Their chemical structure does not obviously resemble that of steroid hormones; hence, their estrogenic effects were totally unexpected. In addition to occupational exposures, environmental estrogens may have played a role in decreasing the quantity and quality of human semen during the last 50 years and in increasing the incidences of testicular cancer and cryptorchidism in men and breast cancer in women and men in industrialized countries. Testing the environmental estrogen hypothesis will require developing appropriate biomarkers of exposure and measuring these biomarkers at developmental points where exposure is critical. We report the ongoing development of a method to extract and separate xenoestrogens from ovarian estrogens with human serum as a source, followed by determination of xenoestrogen concentration by a bioassay. We also critically assess bioassays currently available to measure the cumulative effect of xenoestrogens, e.g., (a) the E-SCREEN assay, which measures the proliferative effect of estrogens on their target cells, and (b) the induction by estrogens of specific gene products, such as progesterone receptor and pS2.

Biological Assay↗
PubMed1995

Variants of the human prostate LNCaP cell line as tools to study discrete components of the androgen-mediated proliferative response.

Androgens regulate the proliferation of their target cells through a sequential two-step mechanism. In the first step, androgens increase proliferation rates; following this proliferative response, a second step of proliferative shutoff ensues. Human prostate LNCaP cell variants were used to assess whether the proliferative and shutoff effects of androgens could be segregated selectively by manipulating the hormonal milieu. The LNCaP-FGC and LNCaP-LNO cell lines were derived from the same biopsy specimen but exhibited different proliferative responses. Cell proliferation was inhibited by treatment with 10% charcoal-dextran stripped human serum in the LNCaP-FGC variant but not in LNCaP-LNO cells. Physiological androgen concentrations induced a proliferative shutoff in LNCaP-LNO cells (G1 arrest), an effect also expressed by LNCaP-FGC cells. This G1 arrest was irreversible in the LNCaP-FGC variant but was reversed upon androgen withdrawal in LNCaP-LNO cells. A new variant (LNCaP-TJA) was selected from LNCaP-LNO cells treated with 30 nM methyltrienolone for 4 months; these cells proliferated maximally regardless of the presence of androgens. All three cell variants had functional androgen receptors, and androgens induced prostate-specific antigen secretion. The androgen-induced proliferative shutoff in LNCaP-FGC and LNO cells was partially antagonized by antiandrogens and was not mediated by autocrine factors. Finally, the variant LNCaP cell lines described herein are probably representative of phenotypes present in prostate cancer patients during the course of this disease and may arise from selective pressure imposed by therapeutic protocols aimed at modifying the hormonal milieu of the host.

Androgen Antagonists↗
PubMed1994

Formation of the male pronucleus, organization of the first interphase monaster, and establishment of a perinuclear plasm domain in the egg of the glossiphoniid leech Theromyzon rude.

Whole-mounted or sectioned eggs of the glossiphoniid leech Theromyzon rude were studied under the dissecting, fluorescence, light, and electron microscope. The egg is often penetrated by a single sperm that enters the animal hemisphere and becomes subjected to migration block. The latter is released shortly before or after discharge of the first pole cell, when the sperm centrosome initiates aster formation, the nucleus begins to be untwisted, and its chromatin decondensed. Sperm centration occurs along one side of the egg and appears to follow an arc-like trajectory as a result of vegetal and inward movements affected by colchicine and cytochalasin B but not by taxol. Results indicate that growing microtubules are needed for both movements, whereas actin filaments are essential for the vegetalward movement only. The sperm centrosome becomes the main microtubule organizing center (MTOC) of the egg and concomitantly originates the elaborate first interphase monaster. Additional peripherally situated MTOC form cytaster-like bodies whose visualization is improved by taxol treatment. A voluminous centrosphere, formed around the sperm centrosome, becomes a center of organelle accumulation, giving rise to a perinuclear plasm domain. This process seems to involve both import and replication of organelles.

Animals↗
PubMed1994

Analysis of risk of death from differentiated thyroid cancer.

The records of 231 patients with differentiated thyroid cancer, treated at the University Hospital of Granada between 1972 and 1986, were reviewed to determine which factors were associated with a favourable response and prolonged survival. Radical surgery was the initial treatment in the large majority of the patients. During the postoperative period, 174 patients received 131I therapy and 12 patients were treated by external irradiation. All of them received hormone replacement therapy. Median follow up was over 5 years. Kaplan-Meier actuarial overall survival (S) and disease-free survival (DSF) at 10 years were used as end points for analysis. Survival and freedom from relapse at this time were 0.93 +/- 0.02 and 0.63 +/- 0.06, respectively. No flattening of the relapse curve was observed during the period of follow-up. Univariate analysis showed that the prognosis was significantly influenced by age, sex (papillary cancer only), histological type of tumour, clinical-pathological stage of disease and cervical lymph node status (entire group and papillary cancer). Using Cox's regression model, two groups of patients with low and moderate risk of death and moderate and high risk of recurrence could be identified.

Actuarial Analysis↗
PubMed1994

Initial radiation-induced DNA damage in human tumour cell lines: a correlation with intrinsic cellular radiosensitivity.

The role of the initial DNA double-strand breaks (dsb) as a determinant of cellular radiosensitivity was studied in human breast and bladder cancer cell lines. Cell survival was measured by monolayer colony-forming assay as appropriate and differences in radiosensitivity were seen (alpha-values ranged from 0.12 to 0.54). After pulsed-field gel electrophoresis (PFGE) the initial slopes of dose-response curves were biphasic with a flattening of the curves above 30 Gy. When the frequency of DNA dsb induction was assessed using a mathematical model based on the DNA fragment size distribution into the gel lane, we found a statistically significant relationship between the number of DNA dsb induced and the corresponding alpha-values and fraction surviving after 2Gy (P = 0.0049 and P = 0.0031 respectively). These results support the view that initial damage is a major determinant of cell radiosensitivity.

Breast Neoplasms↗
PubMed1994

Evaluation of the growth rate of MCF-7 breast cancer multicellular spheroids using three mathematical models.

Growth data on 60 multicellular spheroids of MCF-7 human breast cancer cells were fitted, on an individual basis, by the Gompertz, Bertalanffy and logistic equations. MCF-7 spheroids, initiated and grown in medium containing oestrogens, exhibited a growth rate that decreased continuously as spheroid size increased. Plots of spheroid volume v. time generated sigmoid curves that showed an early portion with an approximately exponential volume increase; a middle region or retardation phase characterized by a continuously decreasing growth rate; and, finally, a late segment or plateau phase approaching zero growth rate, that permitted an estimate of the maximum spheroid size (Vmax). Growth curves generated by MCF-7 spheroids under different experimental conditions (hormones, drugs and radiation exposures) can be compared after normalization. Linearized forms of the fitted Gompertz curves provided a convenient way to express differences in growth rate.

Breast Neoplasms↗
PubMed1992

MCF-7 breast cancer cells grown as multicellular spheroids in vitro: effect of 17 beta-estradiol.

To obtain multicellular spheroids from MCF-7 human breast cancer cells we adhered to the following procedure: (a) limiting the adherence of cell to the substratum; (b) seeding more than the minimum number of cells; (c) guaranteeing the presence of estrogens in the culture medium. Charcoal-dextran (CD)-treated sera seemed to inhibit spheroid formation. A reduction in the concentration of CD-human sera (from 10% to 5%) added to phenol-red-free medium facilitated progress from cellular aggregates to multicellular spheroids. Once the spheroids became initiated, size increased at a rate that showed a good fit to a Gompertzian equation (A = 0.368 +/- 0.067 alpha = 0.065 +/- 0.013, r range = 0.890-0.989). Three different patterns of spheroid morphology and proliferative kinetic were defined: (a) spheroids with diameter less than 200 microns had a constant pattern of heterogeneity in the distribution of 3H-TdR-labelled cells and in the expression of estrogen receptors; (b) spheroids 250 to 700 microns in diameter showed a decrease in the proportion of 3H-TdR-labelled cells accompanying inward progression (50% in the outer shell, less than 10% in a cell layer located at a depth of 150 microns) while, at a depth of 170 microns, of signs of concurrent cellular degeneration and death were apparent; and (c) spheroids with a diameter of greater than 750 microns showed a crust of viable cells uniformly labelled with thymidine without impairment of the proportion of labelled cells when progressing inward from the spheroid crust. The larger the spheroid volume, the lower its growth fraction and the longer its volume doubling time. The hormone-dependence of MCF-7 cells in forming multicellular spheroids represents a unique experimental model for assessing estrogen action on cell organization and proliferation.

Breast Neoplasms↗
PubMed1991

Kinetics of cellular proliferation and hormonal receptors in EVSA-T breast cancer cell line.

The hormone-independent human breast cancer cell line EVSA-T, originally described as negative estrogen and progesterone receptors is shown to become positive hormone receptors when the cellular proliferation rate is slowed down. The experimental procedure included the following steps: 1) EVSA-T cells were seeded in minimum essential medium supplemented with 10% fetal bovine serum and kept undisturbed for 2 days; 2) culture medium was replaced with Dulbecco's solution and Ham's F-12 and cells were incubated in serum-free media for another 24 h; 3) then, cells were "rescued" with 10% FBS supplemented medium and estrogen (ER) and progesterone receptors (PgR) were measured immediately, time 0, and 6, 12, 18, 24 and 30 h after the media were changed. Cell yield was quantified at the same times. Experimental data indicate that changing the proliferation kinetics makes it possible to detect estradiol and progesterone receptors on EVSA-T cells. Estrogen receptor appeared at 18 h after rescue, 6 hours before progesterone receptor could be detected. Immunohistochemical analysis of ER content confirmed this observation, showing maximal positive stain at 18 h. Furthermore, ER disappeared when cells recovered their normal proliferation rate.

Breast Neoplasms↗
PubMed1991

Thyroglobulin level as a predictive factor of tumoral recurrence in differentiated thyroid cancer.

Ninety-eight patients with differentiated thyroid carcinoma were studied. Actuarial methods were used to investigate the 10-yr probability of survival (pS) and disease-free survival (pDFS). Our results show that the pDFS is a function of: (1) clinicopathologic stage: Stages I-II, pDFS = 90.9% +/- 5.0% versus Stages III-IV, pDFS = 55.9% +/- 17.8% (p less than 0.005); (2) age: Age less than 45 yr, pDFS = 87.2% +/- 10.0% versus age greater than or equal to 45 yr, pDFS = 66.6% +/- 12.0% (p less than 0.002); and (3) plasma thyroglobulin (Tg) levels: Tg less than or equal to 23 ng/ml, pDFS = 100% versus Tg greater than 23 ng/ml, pDFS = 68.3% +/- 10.6% (p less than 0.005). Using the multivariate analysis of proportional risk, the regression coefficients obtained (Stage: beta = 0.7615; Age: beta = 1.6398, and Tg: beta = 1.7607) allowed us to establish two different groups of risk of relapse on the basis of a prognostic index.

Adenocarcinoma↗
PubMed1990

Structure and development of the egg of the glossiphoniid leech Theromyzon rude: reorganization of the fertilized egg during completion of the first meiotic division.

Reorganization of the fertilized egg during completion of the first meiotic division was studied in the glossiphoniid leech Theromyzon rude. Rotation of the meiotic spindle, presumably as a result of changes in the length and arrangement of astral fibers, allows one of its poles to approach the prospective animal pole (AP), which appears as a differentiated region of the ectoplasm. The peripheral spindle pole is greatly modified during its anchorage to the AP and is dismantled upon emission of the first pole cell. Meanwhile, the central spindle pole is less modified and is reused during the second meiotic division. Redistribution of microvilli, as well as rearrangement of the ectoplasmic actin lattice, lead to remodeling of the egg surface. Emission of the first pole cell is preceded by a contraction wave that seems to arise by condensation of subcortical actin filaments at the equator of the egg. Poleward displacement of this wave causes evagination of the AP and ooplasmic segregation. A cytokinetic contractile ring forms by assembly of cortical actin filaments at the base of the AP evagination. When this process is disturbed by colchicine or cytochalasin B treatment, abortive or ghost pole cells may be formed.

Animals↗
PubMed1990

The proliferative effect of "anti-androgens" on the androgen-sensitive human prostate tumor cell line LNCaP.

The effect of steroidal and nonsteroidal "anti-androgens" on the proliferative capacity of androgen-sensitive LNCaP-FGC human prostate tumor cells in culture was studied using charcoal-dextran stripped human serum-supplemented media. Cyproterone and medroxyprogesterone acetates, flutamide, hydroxyflutamide, and anandron (R23908) were administered alone at concentrations between 3 X 10(-12) and 3 X 10(-6) M. Results indicated that although medroxyprogesterone induced maximal proliferation at 3 X 10(-9) M, the other "anti-androgens" (with the exception of flutamide that was ineffective) were effective at 3 X 10(-8) M and higher concentrations; the amplitude of the proliferative response by these compounds was comparable to that elicited by estradiol-17 beta (3 to 5-fold over control). None of the anti-androgens tested triggered the shutoff effect characteristic of androgen action. When 3 X 10(-10) M DHT and the above mentioned anti-androgens were administered simultaneously, a synergistic pattern was seen; on the contrary, 3 X 10(-8) M DHT cancelled the proliferative effect of each of the anti-androgens when administered simultaneously. The relative binding affinity of these anti-androgens to androgen receptors present in LNCaP-FGC cells did not correlate well with their proliferative efficiency. The data collected were interpreted within the premises of the negative control hypotheses for the regulation of cell proliferation in metazoans. Within those premises, results became compatible with the notion that first, "anti-androgens" elicited the proliferation of androgen-sensitive cells by neutralizing the effect of a serum-borne inhibitor (androcolyone-I); this event seems not to be mediated by androgens receptors. Second, anti-androgens did not trigger a proliferative shutoff response like androgens do, i.e. the proliferative pattern induced by anti-androgens was comparable to that elicited by estrogens and progestins. Third, when administered simultaneously with 3 X 10(-10) M DHT, anti-androgens behaved synergistically. Fourth, the DHT-induced shutoff effect consistently overrode the proliferative effect generated by anti-androgens and estrogens when added alone. Finally, taken together these results raise important questions regarding the therapeutic role of anti-androgens in prostate cancer.

Androgens↗
PubMed1990

Relationship between proliferative activity and cellular hormono-dependence in the MCF-7 breast cancer cell line.

Research on kinetic and hormonal features of breast cancer has led to the development of indices which either reflect accurately the prognosis (incorporation of tritium labelled thymidine) or predict the response to hormonal treatment (presence and concentration of estrogen and progesterone receptors). However, the relationship between cellular proliferation and tumour hormono-dependence has been little studied so far. We describe this relationship in the hormone-dependent MCF-7 cell line cultured in monolayers in MEM + 10% FCS or MEM + 10% FCS (s). We have found that: 1) cellular proliferation and estrogen or progesterone receptor concentration were mutually dependent, the greatest estradiol binding capacity was obtained in cells in which mitotic activity had been slowed down (G0/G1) by the antiestrogenic action of hydroxytamoxifen added to the culture; 2) the presence of estradiol in the culture medium induced marked changes in the synthesis and catabolism of estrogen and progesterone receptors; and 3) both receptors acted as functional proteins whose intracellular concentrations varied depending on the phases of the mitotic cycle.

Breast Neoplasms↗
PubMed1990

[Hormone dependence in breast and prostate tumor cell lines].

Knowledge about the role of sex hormones in the control of cell proliferation and cell-type specific protein synthesis is mainly collected by using cell culture techniques. The adoption of cell culture models addressed at defining these issues is due to the uncomplicated assessment of reliable proliferation-related parameters. Established cell lines derived from estrogen and androgen sensitive tissues, have been used in proliferation studies for more than thirty years. The data gathered so far can be summarized in three following working hypotheses: the direct and indirect-positive hypotheses and the indirect-negative hypothesis. Further characterization and assessment of the hormone dependence of growth factors and growth inhibitors will allow for the mechanistic understanding of the regulation of cell proliferation by sex hormones.

Androgens↗
PubMed1990

[Tissue polypeptide antigen liberation and proliferation of MCF-7 cells after synchronization with hydroxytamoxifen and rescue with estradiol].

The kinetic of release of the tissular polypeptide antigen (TPA) by MCF-7 cells synchronised by sequential treatment with hydroxytamoxifen (OH-TAM) and 17 beta-estradiol has been studied. The present findings confirm the proliferative effect of estradiol on MCF-7 cells, with a shortening of the doubling time (TD) (22.2 h versus 24.8 h) and an increase in the growing fraction (Fc) (94% versus 81%) when compared with the same parameters measured in cells rescued from OH-TAM but not treated with estradiol. In addition, the action of estradiol was followed by a simultaneous increase in the amount of TPA in the culture medium related with the phases G2/M and G1 of the mitotic cycle. This phenomenon seems to be the reason for the steplike shape of the TPA released curves. The experimental results suggest that in MCF-7 cells the sequential combination of antiestrogenic agents and estrogens leads to a cellular synchronization. Furthermore this synchronization is maintained for at least 3 cycles of cell division.

Antigens↗