Ezrin has a COOH-terminal actin-binding site that is conserved in the ezrin protein family.

Ezrin, previously also known as cytovillin, p81, and 80K, is a cytoplasmic protein enriched in microvilli and other cell surface structures. Ezrin is postulated to have a membrane-cytoskeleton linker role. Recent findings have also revealed that the NH2-terminal domain of ezrin is associated with the plasma membrane and the COOH-terminal domain with the cytoskeleton (Algrain, M., O. Turunen, A. Vaheri, D. Louvard, and M. Arpin. 1993. J. Cell Biol. 120: 129-139). Using bacterially expressed fragments of ezrin we now demonstrate that ezrin has an actin-binding capability. We used glutathione-S-transferase fusion proteins of truncated ezrin in affinity chromatography to bind actin from the cell extract or purified rabbit muscle actin. We detected a binding site for filamentous actin that was localized to the COOH-terminal 34 amino acids of ezrin. No binding of monomeric actin was detected in the assay. The region corresponding to the COOH-terminal actin-binding site in ezrin is highly conserved in moesin, actin-capping protein radixin and EM10 protein of E. multilocularis, but not in merlin/schwannomin. Consequently, this site is a potential actin-binding site also in the other members of the protein family. Furthermore, the actin-binding site in ezrin shows sequence homology to the actin-binding site in the COOH terminus of the beta subunit of the actin-capping protein CapZ and one of the potential actin-binding sites in myosin heavy chain. The actin-binding capability of ezrin supports its proposed role as a membrane-cytoskeleton linker.

Identification of recognition sites for myc/max/mxd network proteins by a whole human chromosome 19 selection strategy.

In this study, we have identified 20 human sequences containing Myc network binding sites in a library from the whole human chromosome 19. We demonstrated binding of the Max protein to these sequences both in vitro and in vivo. The majority of the identified sequences contained one or several CACGTG or CATGTG E-boxes. Several of these sites were located within introns or in their vicinity and the corresponding genes were found to be up- or down-regulated in differentiating HL-60 cells. Our data show the proof of principle for using this strategy in identification of Max target genes, and this method can also be applied for other transcription factors.

Presence of alpha-2-macroglobulin in normal but not in malignant cervical epithelium.

alpha-2-Macroglobulin (alpha 2M) was demonstrated in normal and mildly dysplastic cervical epithelium using immunohistochemical methods. In contrast, staining for alpha 2M diminished gradually with advancing epithelial dysplasia and was totally negative in truly neoplastic lesions. No in vitro synthesis could be detected in cultured ectocervical cells or in a corresponding malignant cell line (HeLa). However, alpha 2M was taken up from added human serum by cultured normal ectocervical cells, whereas HeLa cells remained negative also under these conditions. These and previous results suggest that normal cells may have the capacity to synthesize or endocytose alpha 2M, thus possibly regulating their pericellular proteolysis, while these functions may be defective in malignantly transformed cells.

Long-term transplantability and morphological stability of three experimentally induced urinary bladder carcinomas in rats.

Three transitional cell carcinomas induced in Fischer 344 rats by a methylcholanthrene pellet or a foreign body inserted locally into the bladder have been serially transplanted in the syngeneic strain for up to 6.5 years. There have been no changes in the individual morphological characteristics of the tumors during this time. Cells cultured in vitro for varying numbers of passages reproduce regularly the morphology of each tumor when they are injected back into the animals and results from a microcytotoxicity assay for cellular immunity indicate that they retain a common, bladder tumor-specific antigen. These tumors are useful for research in turmo biology and are offered to other scientists seeking transplantable carcinomas for experimentation.

Distinct chromosomal imbalances in uterine serous and endometrioid carcinomas.

Endometrial carcinoma shows various histological types that differ in their clinical presentation and prognosis. Comparative genomic hybridization was used to detect gains and losses of DNA sequences along all chromosome arms in 24 uterine serous and 24 uterine endometrioid carcinomas. In serous carcinomas, extensive genetic aberrations were detected in 17 of the 24 specimens, with a mean of 5.7 changes per tumor. The most frequent gains occurred at 3q (50%), 8q (33%), 5p (29%), 6p (29%), and 1q (29%), and the most common losses were located at 4q (17%), 15q (17%), and 18q (17%). Tumors exhibiting DNA copy number changes were associated with shorter overall survival. In endometrioid carcinomas, genetic aberrations were less frequent and simpler than in serous carcinomas. DNA sequence copy number changes were observed in 12 of the 24 cases, with a mean of 1.5 changes per tumor. The most frequent aberrations were gains at 1q (29%), 2q (13%), and 8q (13%). Losses were rarely observed. The diverging pattern of genetic changes observed in uterine serous and endometrioid carcinomas suggests different pathways of carcinogenesis in these tumor types.

Presence of alpha 2-macroglobulin in normal but not in malignant human syncytiotrophoblasts.

alpha 2-Macroglobulin (alpha 2M) was demonstrated in normal syncytiotrophoblasts of both early and full-term human placentas using immunocytological staining. alpha 2M was also detected in hydatidiform moles, the benign tumors of proliferating syncytiotrophoblasts. In contrast, no alpha 2M was detected in invasive moles or choriocarcinomas. In culture conditions, both normal syncytiotrophoblasts and choriocarcinoma cells, identified by production of human chorionic gonadotropin, were negative when stained for alpha 2M or when studied using metabolic labeling and immunoprecipitation or radioimmunoassay. However, alpha 2M was taken up from added human serum by the cultured syncytiotrophoblasts, whereas choriocarcinoma cells remained negative also under these conditions. The possible role of alpha 2M in the regulation of proteolysis in cell invasion is considered.

Redistribution of Mr 75,000 plasma membrane protein, cytovillin, into newly formed microvilli in herpes simplex and Semliki Forest virus infected human embryonal fibroblasts.

We have previously purified an Mr 75,000 protein from cultured human JEG-3 choriocarcinoma cells and showed that this protein is specifically confined to the cytoplasmic side of JEG-3 microvillar membranes. Recently, the Mr 75,000 protein, designated as cytovillin, was found to be expressed also in several other cultured human cell lines and strains, in which it was detected in microvillus-related structures. We now demonstrate the redistribution of cytovillin in herpes simplex type 1 (HSV-1) and Semliki Forest virus (SFV) infected human embryonal fibroblasts. Virus infection induced rapidly numerous microvilli on the apical cell surfaces, and cytovillin was enriched into these newly formed structures as shown by indirect immunofluorescence and immunoferritin electron microscopy. In mock-infected cells treated with the anti-cytovillin antibodies a small amount of ferritin particles and faint fluorescence was detected along the smooth plasma membrane. Only occasional cell surface protrusions were observed in these cells. The enrichment of the cytovillin was first seen 2 h after infection. The isoelectric point (IP) and the mobility of the cytovillin polypeptide in sodium dodecyl sulfate polyacrylamide gel electrophoresis was not altered after this redistribution, suggesting that the protein was not significantly modified during infection. Five RNA+ SFV mutants (ts-1, ts-2, ts-3, ts-5, ts-7) with temperature-sensitive defects in processing and transport of viral envelope glycoproteins to the plasma membrane induced microvilli at the restrictive temperature (39 degrees C) as the wild type virus.(ABSTRACT TRUNCATED AT 250 WORDS)

Estrogen receptor beta in breast cancer.

Estrogen is essential for normal growth and differentiation in the mammary gland. It also supports growth of approximately 50% of primary breast cancers. For this reason, removal of estrogen or blocking of its action with the anti-estrogen, tamoxifen, is the main treatment for estrogen receptor alpha (ERalpha)-positive tumors. In 1996, when oncologists became aware of a second ER, ERbeta, there was some doubt as to whether this receptor would be of importance in breast cancer because the clinical consensus was that responsiveness to tamoxifen is related to the presence of ERalpha in breast cancer. Today we know that ERalpha and ERbeta have distinct cellular distributions, regulate separate sets of genes and can oppose each other's actions on some genes. We also know that ERbeta is widely expressed in both the normal and malignant breast and that there are proliferating cells in the breast which express ERbeta. In this review we summarize what is known about ERbeta in breast cancer and examine the possibility that ERbeta-selective ligands may well represent a useful class of pharmacological tools with a novel target, namely proliferating cells expressing ERbeta.

Synthesis of placental protein 12 by human decidua.

The synthesis and secretion of placental protein 12 (PP12) were studied in tissue culture using explants of decidua, amnion, chorion, and placenta from seven full term pregnancies. The total amounts of PP12 in media and tissues were measured by RIA, and new protein synthesis and secretion by decidual explants were demonstrated by the incorporation of [35S]methionine into PP12 after 20 h of incubation with 12.5 microCi/ml [35S]methionine. Cycloheximide was used to study the effect of a protein synthesis inhibitor on the secretion of PP12 by decidua. Significantly more PP12 (P less than 0.001) was released into the medium from decidual explants than from chorion and amnion explants throughout the experimental period of 24 h. When incubated under identical conditions, placental explants released no detectable PP12. In decidual tissues and their culture media, the total amount of PP12 was 127.4% higher after incubation than before incubation (P less than 0.001). No increase was found when chorion and amnion were cultured. The addition of cycloheximide into cultures decreased the total amount of PP12 in the decidua and in its culture medium by more than 50%, indicating that one part of PP12 in decidua was performed and another part was newly synthesized. Decidual explants incorporated [35S]methionine into immunoprecipitable PP12 indicating new PP12 synthesis. In gel filtration, 77% of decidual [35S] PP12 eluted in the same position as purified PP12. In sodium dodecyl sulfate polyacrylamide gel electrophoresis, the migration mobility of [35S]PP12 was identical with that of purified PP12. Our results clearly demonstrate that PP12 is a decidual rather than a placental protein.

Secretory endometrium synthesizes placental protein 14.

Saline extracts of human nonpregnant endometrium were found to contain placental protein 14 (PP14). The tissue PP14 content was highest in the late secretory phase (median, 7.7 mg/g protein; n = 14), whereas proliferative endometrium (n = 8) was either PP14 negative or showed a low PP14 content (median, 0.15 mg/g protein). By immunoperoxidase staining, PP14 was localized in the glandular epithelial cells of endometrium. In tissue culture, secretory endometrium released more PP14 than proliferative endometrium, and cycloheximide markedly decreased this release. Synthesis of PP14 by secretory endometrium was demonstrated by incorporation of [35S]methionine into immunoprecipitable PP14. These results show that PP14 is synthesized and secreted by the nonpregnant endometrium.

Synthesis of placental protein 12 by human endometrium.

We have previously shown that placental protein 12 (PP12) is synthesized and secreted by human term pregnancy decidua in vitro. In the present study, fragments of proliferative and secretory phase endometrium were cultured in media in the presence and absence of progesterone (P) and 17 beta-estradiol (E2) for 96 h. The PP12 concentrations in the media and tissues were measured by RIA, and de novo synthesis was investigated by measuring the incorporation of [35S]methionine into PP12. Before culture, PP12 could not be detected in any proliferative endometria, whereas all secretory endometria contained PP12. All secretory endometria released PP12 into the medium in the presence and absence of added P and E2. Secretory endometria released significantly more PP12 than proliferative endometria. Three of seven proliferative endometria did not release PP12 in the absence of P, but all did so after P had been added. The addition of P to culture medium caused a 2.4-to over 71-fold increase in PP12 secretion over control values in proliferative endometria and up to a 3.5-fold increase in secretory endometrium. E2 had no significant effect. Cycloheximide totally inhibited the PP12 release induced by P from proliferative endometrium, and in secretory endometrium, it either totally blocked PP12 release or inhibited the stimulation due to P. [35S]Methionine was incorporated into immunoprecipitable PP12 in cultures of secretory and P-treated proliferative phase endometria. These results demonstrate de novo synthesis of PP12 by nonpregnant endometrium in tissue culture and suggest that the biosynthesis and secretion of PP12 by nonpregnant endometrium are regulated by P.

Immunological evidence for the occurrence of luteinizing hormone-releasing factor and the alpha-subunit of glycoprotein hormones in carcinoid tumors.

The immunoperoxidase technique was used to identify LRF and glycoprotein hormones or their subunits in 22 carcinoid tumors of various origins. All tumors were positive for both LRF and the alpha-subunit, whereas stainings with antisera against the beta-subunits of CG, LH, FSH, and TSH were negative. The binding of LRF and the alpha-subunit within the same malignant tissue raises the possibility of a functional relationship between the two.

Pregnancy-associated plasma protein A in the human endometrium is dependent on the effect of progesterone.

The biotin-avidin immunoperoxidase method was used to study pregnancy-associated plasma protein A (PAPP-A) in the endometrium of 102 pre- or postmenopausal women. Endometria in the proliferative phase (n = 27) or early secretory phase (days 1-3 postovulation; n = 12), cystic glandular hyperplasia (n = 12), and postmenopausal endometria of untreated (n = 7) or estrogen-treated (n = 5) women were all PAPP-A negative. By contrast, PAPP-A was invariably present in the endometrium during the normal secretory phase after day 3 of ovulation (n = 28), during cyclic progestogen treatment of pre-menopausal women (n = 6), and during estrogen-progestogen replacement therapy of postmenopausal women (n = 5). These results suggest that the occurrence of PAPP-A in the endometrium is progesterone dependent.

Localization of luteinizing hormone, follicle-stimulating hormone, prolactin, and their receptors in human and rat testis using immunohistochemistry and radioreceptor assay.

Immunoperoxidase staining and radioreceptor assay were used to study the localization of LH, FSH, and PRL and their receptors in the human and rat testis. In immunohistochemical staining, the Leydig cells of both species were invariably LH positive and generally FSH negative, but there were some FSH positive cells which were morphologically indistinguishable from the Leydig cells. The tubules were LH negative. The Sertoli cells of both species were FSH positive, whereas the spermatogonia and other germ cells were negative. Positive staining for PRL was seen in rat Leydig cells, whereas the human testes were negative. In keeping with the immunohistochemical findings, LH and FSH receptors were found in the testis of both species, but PRL receptors only in the rat. The finding of FSH positive cells in the interstitial tissue may explain why FSH increases the number of Leydig cell LH receptors and increases the sensitivity and maximum response to LH stimulation. Failure to demonstrate PRL and PRL receptors in the human testis indicates either that very low receptor concentrations are needed to bring about PRL action or that the established testicular effects of PRL are indirect.

Immunohistochemical demonstration of relaxin in the genital tract of pregnant and nonpregnant women.

The biotin-avidin immunoperoxidase staining method and antisera against highly purified porcine relaxin were used to localize relaxin in the genital tract of pregnant and nonpregnant women. Formalin-fixed tissue specimens from normal placenta, decidua, myometrium, vagina, corpus luteum, and Fallopian tubes were studied. In pregnant women, relaxin was found in the placental syncytiotrophoblast, decidua, and corpus luteum. In nonpregnant women, relaxin was identified in the corpus luteum and endometrium in the secretory, but not in the proliferative, phase. Myometrium, cervix, vagina, and Fallopian tubes were negative for relaxin. This is the first report describing relaxin in the nonpregnant corpus luteum, and we also confirm results of an early disputed study claiming that endometrium in the secretory phase contains relaxin. The origin and biological role of human endometrial relaxin remain to be studied.

Evidence for the presence of oncoplacental SP1-like protein in normal nonpregnant serum.

To seek the pregnancy-specific beta 1-glycoprotein (SP1) in nonpregnant serum, normal human serum was applied to immunoadsorbent containing monoclonal anti-SP1 antibodies. SP1 eluted with 8 M urea was further analyzed by sodium dodecyl sulfate-gel electrophoresis and immunoblotting. A SP1-positive band with the same electrophoretic mobility as purified placental SP1 was found. The results suggest that serum from normal nonpregnant subjects contains material closely related to the placental protein SP1. The mean serum concentrations of SP1 were similar in men and women, ranging from 1.1-3.4 ng/ml.

Pregnancy-specific beta-1-glycoprotein (SP1) in cultured amniotic fluid cells.

The synthesis of pregnancy-specific beta-1-glycoprotein (SP1) was studied in amniotic fluid cell cultures using RIA, immunoperoxidase, and immunofluorescence techniques. SP1 was found by RIA in all 11 sonicates and in 21 of 26 culture media. The SP1-immunoreactive material was immunologically similar to maternal serum SP1. Immunoperoxidase and indirect immunofluorescence staining were positive in large cells identified as epithelial amniotic cells by labeling with antikeratin antibodies. Fibroblast-like cells were occasionally found in cultures, but they did not contain demonstrable amounts of SP1. The physiological significance of the findings presented remains unclear.

Pregnancy-specific beta 1-glycoprotein-like material in human cerebrospinal fluid.

Human cerebrospinal fluid (CSF) from 34 unselected neurological patients was studied for pregnancy-specific beta 1-glycoprotein (SP1) activity because of the recent finding of SP1 production by cultured glial cells. An organic central nervous system lesion was diagnosed in 9 patients, but not in the other 25. Low levels of SP1 immunoreactivity were found in CSF by RIA, and the adsorption of anti-SP1 antiserum with concentrated CSF abolished the positive immunohistochemical staining of placental tissue obtained with the unadsorbed antiserum. By means of immunoadsorption using monoclonal anti-SP1 antibodies, it was possible to isolate SP1 immunoreactive material from CSF and to demonstrate that it had the same electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis as purified placental SP1. These results show that CSF contains SP1-like material that is closely related, if not identical, to placental SP1. The amount of SP1 in CSF has no direct correlation to an organic central nervous system lesion or to abnormality of the CSF.

Human preovulatory follicular fluid, luteinized cells of hyperstimulated preovulatory follicles, and corpus luteum contain placental protein 12.

RIA gel filtration, isoelectric focusing; and immunoperoxidase staining were employed to study the occurrence and physicochemical characteristics of placental protein 12 (PP12) in the human ovary, corpus luteum, and preovulatory follicular fluid. Fluid aspirated from 75 follicles from 22 women hyperstimulated for in vitro fertilization contained 6-230 micrograms/liter PP12-like immunoreactive material. The dose-response curves of follicular fluid PP12, amniotic fluid PP12, and purified human placental PP12 were parallel in the PP12 RIA. In gel filtration, follicular fluid PP12 eluted in the same volume as purified PP12. The isoelectric point of follicular fluid PP12 was 4.9 and that of purified placental PP12 4.6-4.7. A positive correlation was found between follicular fluid estradiol and PP12, progesterone and PP12, and follicular fluid volume and PP12 concentrations. By immunoperoxidase staining, PP12 was not detectable in unstimulated ovarian tissue before ovulation. In hyperstimulated preovulatory follicles biopsied in connection with follicle aspiration, PP12 was found in the granulosa cells which were luteinized (n = 3), whereas in those hyperstimulated follicles (n = 5) with no luteinization, no PP12 was found either. PP12 was seen in all corpora lutea (n = 5) from unstimulated menstrual cycles. These results show that the occurrence of PP12 is not limited to the placenta. The correlation between follicular fluid steroid and PP12 levels and the findings by immunoperoxidase staining suggest that PP12 is related to endocrine phenomena of the ovary, possibly to the luteinization process.

A novel method to culture laryngeal human papillomavirus-positive epithelial cells produces papilloma-type cytology on collagen rafts.

A novel method to culture human papillomavirus (HPV) positive laryngeal epithelial cells is described. Biopsies of laryngeal papillomas and of HPV-positive laryngeal mucosa were first cultured as a monolayer in which irradiated laryngeal fibroblasts originally derived from a papilloma (PPLF-XR) patient served as feeder cells. When these fourth or fifth passage epithelial cells were transferred to allow growth on an organotypic growth base (collagen raft containing unirradiated PPLF), they grew as a multilayer. This layer showed features typical of HPV infection with koilocytosis, parakeratosis, and isolated dyskeratotic cells. Based on in situ hybridisation, the original tumour sections and epithelial cells from each monolayer passage, as well as the collagen raft sections, contained HPV DNA. Our results show that HPV-infected epithelial cells can be maintained during passages in monolayer culture and that PPLF can support the growth of these cells well. The monolayer cell culture and the collagen raft, the latter providing differentiation-promoting effects, appears to facilitate maintenance of the infected cells and of the viral genome.