Loss of ovarian function and the risk of ovarian cancer

Animal models with premature ovarian failure resulting from the loss or depletion of germ cells consistently develop ovarian surface epithelial cell hyperplasia with invasion into the stroma and the development of ovarian tubular adenomas. In human ovaries, deep epithelial invaginations and inclusion cysts occur at increasing frequency with age and are thought to be the structures from which the majority of ovarian cancers arise. A feature that is common to these animal models and to post-menopausal women is a deficiency in the number of oocytes. The potential consequences of the loss or depletion of female germ cells, naturally or otherwise, include failure of follicle development, significant reductions in oestrogen and progesterone levels and elevation of circulating levels of gonadotropins. This review will consider the way in which these structural and hormonal changes affect ovarian cancer risk. Some lessons may be learned from gonad formation, since notable similarities exist between ovarian tumorigenesis and embryonic gonadogenesis including fragmentation of the basement membrane underlying the coelomic (surface) epithelium, the potential for the migration of epithelial cells into the gonad and the importance of the germ cells for the regulation of ovarian structure and function. Research was supported by grants from the National Cancer Institute of Canada and the Canadian Institutes of Health Research.     

Methods for the study ofin vitro immunization of mouse spleen cells

Conclusion We have reviewed some of our experiences in developing techniques for studying the functions of the cells of the immune system. It is quite clear that much remains to be done. Improvements in the culture system are needed to permit cells to be grown for longer durations and at lower cell concentrations. The important effects of fetal calf serum should be defined. More sophisticated methods for separating cells into distinct functional populations must be developed. New assays for identifying other functions of the cells, particularly a method for directly assaying the number of precursor cells in a population, are needed. When these techniques are applied to the study of immune cells, further facts should be learned which will permit the development of significant, testable hypotheses on the function and relationships of the cells of the immune system. This is publication No. 298 from the Department of Experimental Pathology, Scripps Clinic and Research Foundation, La Jolla, California 92037. This work was supported in part by U.S.P.H.S. Grant 7007 and in part by American Cancer Society Grant E-395. Dr. Mishell is supported by American Cancer Society Grant E-395. Dr. Dutton is supported by a Dernham Fellowship of the California Division, American Cancer Society (No. D-100). Dr. Raidt is supported by United States Public Health Service Postdoctoral Fellowship No. 7-F2-A1-31,590.     

Organization and signaling of endothelial cell-to-cell junctions in various regions of the blood and lymphatic vascular trees

Adhesive intercellular junctions between endothelial cells are formed by tight junctions and adherens junctions. In addition to promoting cell-to-cell adhesion, these structures regulate paracellular permeability, contact inhibition of endothelial cell growth, cell survival, and maintenance of cell polarity. Furthermore, adherens junctions are required for the correct organization of new vessels during embryo development or during tissue proliferation in the adult. Extensive research on cultured epithelial and endothelial cells has resulted in the identification of many molecular components of tight junctions and adherens junctions. Such studies have revealed the complexity of these structures, which are formed by membrane-associated adhesion proteins and a network of several intracellular signaling partners. This review focuses on the structural organization of junctional structures and their functional interactions in the endothelium of blood vessels and lymphatics. We emphasize the way that these structures regulate endothelial cell homeostasis by transferring specific intracellular signals and by modulating activation and signaling of growth factor receptors. This work was supported by the Associazione Italiana per la Ricerca sul Cancro, Association for International Cancer Research, European Community (Integrated Project Contract no. LSHG-CT-2004–503573; NoE MAIN 502935; NoE EVGN 503254; EUSTROKE consortium; Angioscaff consortium; Optistem consortium), Istituto Superiore di Sanità, Italian Ministry of Health, MIUR (COFIN prot: 2006058482_002), and Fondation Leducq Transatlantic Network of Excellence (E.D.). Additional support came from US National Institutes of Health grants HL24136 and HL59157 from the National Heart, Lung, and Blood Institute and CA82923 from the National Cancer Institute and AngelWorks Foundation (D.McD.).     

Immunocytochemical localization of choline acetyltransferase and muscarinic ACh receptors in the antenna during development of the sphinx moth <Emphasis Type="Italic">Manduca sexta</Emphasis>

Immunocytochemistry with monoclonal antibodies was used to investigate the locations of muscarinic acetylcholine receptors (mAChR) and choline acetyltransferase (ChAT) in sections of the developing antennae of the moth Manduca sexta. The results were correlated with a previous morphological investigation in the developing antennae which allowed us to locate different cell types at various stages of development. Our findings indicated that the muscarinic cholinergic system was not restricted to the sensory neurons but was also present in glial and epidermal cells. By day 4–5 of adult development, immunoreactivity against both antibodies was present in the axons of the antennal nerve, and more intense labeling was present in sections from older pupae. At days 4–9, the cell bodies of the sensory neurons in the basal part of the epidermis were also intensely immunolabeled by the anti-mAChR antibody. In mature flagella, large numbers of cells, some with processes into hairs, were strongly labeled by both antibodies. Antennal glial cells were intensely immunolabeled with both antibodies by days 4–5, but in later stages, it was not possible to discriminate between glial and neural staining. At days 4–9, we observed a distinctly labeled layer of epidermal cells close to the developing cuticle. The expression of both ChAT and mAChRs by neurons in moth antennae may allow the regulation of excitability by endogenous ACh. Cholinergic communication between neurons and glia may be part of the system that guides axon elongation during development. The cholinergic system in the apical part of the developing epidermis could be involved in cuticle formation.This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada (NSERC), the Canadian Foundation for Innovation, and the Nova Scotia Research and Innovation Trust to P.H.T. and a NSERC postdoctoral fellowship to J.C.     

Lack of evidence for activation of a serum factor in protease-induced differentiation of mouse erythroleukemia cells

summary The addition of certain proteases to cultures of Friend virus-infected mouse erythroleukemia cells can induced up to 90% of the cells in culture to become hemoglobin-containing, as assessed by positive staining for benzidine (B⁺). Because the mechanism of this protease action is unknown, media components were studied as possible targets for protease activity. Aliquots of medium plus serum were incubated for various times with levels of protease sufficient to induce approximately 50% of the cells to the B⁺ state. Cells were added to protease-pretreated serum either before or after inactivation of the protease. In all cases, enzymatically active protease had to be present with the cells to induce B⁺ cells to form. Serum and other components of the medium pretreated with protease were inactive. Mouse erythroleukemia cells grown in the absence of serum were also induced by proteases to form B⁺ cells. These data imply that the inducing action of proteases cannot be passively transferred by protease-pretreated serum or medium nor is serum required for protease-mediated induction of B⁺ cells. Taken together, these conclusions suggest that the protease action is on the cells or on cellular products intimately associated with cells. This study was supported by U.S. Public Health Service Grants CA 24403 and CA 37874; American Cancer Society Grant CH-303; the Spingold Foundation; the Chemotherapy Foundation, Inc; the Gar Reichman Foundation; an Irving Alpert Cancer Research Award; and institutional general research funds. Part of this work was presented at the 1984 meeting of the American Society of Biological Chemists and American Association of Immunologists, St. Louis, MO (21).     

Autoradiographic and microspectrophotometric studies of DNA synthesis in excised tobacco pith tissue

Summary Pith tissue was cultured on modified White’s nutrient medium supplemented, except for controls, with 2 mg/l of IAA and/or 0,5 mg/l of kinetin. For autoradiographs sections were used from tissue grown on medium containing tritiated thymidine. Nuclear DNA contents (Feulgen) were measured by the microspectrophotometric two-wavelengths method. No fading of Feulgen dye in nuclei was found in 11 weeks, in contrast to considerable fading observed in earlier work when a different batch of basic fuchsin had been employed. Counts of radioactive nuclei in autoradiographs agreed well with microspectrophotometric results on the occurrance of DNA synthesis. In control cultures, with or without tritiated thymidine, DNA doubling took place in about 20% of the nuclei during the first two days but in few, if any, thereafter. It was confirmed that kinetin, as well as IAA, increases the frequency of nuclei undergoing DNA synthesis. However, IAA, in contrast with kinetin, still induced considerable DNA doubling after two days. Continued cell reproduction was maintained only in the presence of both substances. This work has been supported in part by research grants toK. Patau from the US Public Health Service (grant No. C-3313) and the American Cancer Society; and by grants toF. Skoog from the American Cancer Society and the Research Committee of the University of Wisconsin Graduate School with funds from the Wisconsin Alumni Research Foundation.     

Quantitation of human mammary epithelial antigens in cells cultured from normal and cancerous breast tissues

Summary A sensitive radioimmunoassay technique was developed to quantitatite the level of human breast celltype specific antigens on cells from normal breast and from various established cell lines of breast and nonbreast origins. Polyacrylamide gel electrophoresis revealed four major proteinaceous components (150,000; 75,000; 60,000; and 48,000) in human milk fat globule membranes that were used to immunize rabbits in order to elicit antimammary epithelial cell antibody. Antisera obtained were rendered specific by abosorptions and were able to recognize three specific mammary epithelial components of the breast epithelial cell. Human mammary epithelial (HME) antigen expression was highest (1290 ng/10⁶ cells) in normal breast epithelial cells from primary cultures of normal breasts. Lower levels (range: 955 to 330 ng/10⁶ cells) were found in breast epithelial cells from cell lines established from cancerous breast tissue. Cells of nonbreast origins as well as fibroblasts from breast gave much lower values (less than 30 ng/10⁶ cells). On treatment, with trypsin, of two breast epithelial cell lines (MDA-MB-157 and MCF-7) 80 to 85% of their HME antigen expression was lost, suggesting that a majority of these breast antigens reside on the cell surface. This work was Supported by Grant PTD-99 from the American Cancer Society, Grant CA19455 and CA20286 from the National Cancer Institute, and Biomedical Research Support Grant RR05467 from the National Institutes of Health. Most cells used in the present study were produced with support from National Cancer Institute Contract Y01-CP8-0500, Biological Carcinogenesis Branch, Division of Cancer Cause and Prevention, under the auspices of the Office of Naval Research and the Regents of the University of California.     

Lack of gp120-induced anergy and apoptosis in chimpanzees is correlated with resistance to AIDS

Human immunodeficiency virus-1 (HIV-1) infects both humans and chimpanzees, but in the chimpanzee, HIV-1 infection leads only very rarely to loss of CD4 T cells or to AIDS-like disease. The pathogenetic basis for this difference in host range is not understood. In previous studies, using CD4 T cells from HIV-1 seronegative human donors, we demonstrated that crosslinking of CD4-bound gp120, followed by signaling through the T cell receptor for antigen (TCR), resulted in cell death by apoptosis. To determine whether activation-induced apoptosis correlates with progression to AIDS, we studied the chimpanzee. Our data suggest that, although human CD4 T cells respond to CD4 ligation with anergy and apoptosis upon activation, chimpanzee CD4 T cells do not undergo apoptosis after cross-linking of CD4-bound gp120, followed by signaling through the TCR. In addition, proliferation assays show that chimpanzee CD4 T cells do not become anergic after CD4 ligation. Thus, it is possible that, in the chimpanzee, the absence of cellular anergy and apoptotic cell death after CD4 ligation by HIV-1 gp120 protect this primate species from progression to AIDS-like disease.This investigation was supported by National Institute of Health grants AI-30575, AI-29903, AI-35513, and RR00015 (TH Finkel), AI-05060 (WC Satterfield), American Foundation for AIDS Research grants 02270-16-RG (TH Finkel) and 770188-11-PF (NK Banda), the Concerned Parents for AIDS Research, the UCHSC Cancer Center, the Eleanore and Michael Stobin Trust, and the Bender Foundation.     

Growth of normal human mammary cells in culture

Summary Reduction mammoplasty tissue was used to obtain short-term cultures of human epithelial cell populations. Digestion of tissue with collagenase and hyaluronidase resulted in cell clusters (organoids) resembling ductal and alveolar structures; these could be separated by filtration from the stromal components. Epithelial outgrowth from these organoids was greatly enhanced by the addition of conditioned medium from other human epithelial and myoepithelial cell lines. Additionally, the mammary epithelial growth was stimulated by insulin, hydrocortisone, epidermal growth factor, and steroid hormones. With this enriched nutritional environment, active cell division could be maintained for 1 to 3 months and cells could be serially subcultured 1 to 4 times. This research was supported by Grant PDT-72 from the American Cancer Society and Grant CP-70510 from the National Institutes of Health.     

Development,characterization, and viral susceptibility of a feline (Felis catus) renal cell line (CRFK)

Summary Cell line CRFK, derived from kidney tissue of a normal domestic kitten, was initiated in 1964. With intermittent periods of storage in the frozen state, it has been grown in vitro during more than 200 passages, without apparent loss of susceptibility to selected viruses. Various herpesviruses and feline viruses belonging to differnet virus groups grow readily and with distinct, cytopathic features. The cells now grow as a smooth monolayer of epithelial-like cells; most have 37 chromosomes (2n−1) and are thus aneuploid for cat karyotype. Three distinct marker chromosomes are identified. The cell line, which is free of mycoplasmal contaimination, is useful in feline virus research and diagnostic medicine and has become of particular interest in cancer research. Supported in part by Contract E73-2001-NO1-CP-3-3237 within the Special Virus Cancer Program, National Cancer Institute, National Institutes of Health, Public Health Service and the Morris Animal Foundation and General Research Support funds of the New York State Veterinary College. CRFK cells from stock provided by C. G. Fabricant are available for distribution to investigators from the Cell Culture Laboratory, Naval Biomedical Research Laboratory.     

Monolayer culture of human endometrium: Methods of culture and identification of cell types

Summary Monolayer cultures can be established from human endometrial tissue after enzymatic dispersal into isolated glands or single cells. Three cell types that have distinct morphology by light and electron microscopy are observed in the resulting primary cultures. One cell type, an elongated spindle cell, is similar in appearance to fibroblasts derived from other tissues. A second cell type forms colonies of tightly cohesive cells, ranging in shape from oval to polygonal. These cells have typical organelles and junctional complexes characteristic of epithelial cells from the endometrium. The third cell type assumes a pavement-like appearance composed of polygonal cells when viewed by phase contrast microscopy, but lacks distinctive ultrastructural features of epithelial cells. These cells in culture resemble the endometrial stromal cell, the predominant cell type of the human endometrium in vivo. The epithelial cell does not survive subculturing but the other two cell types can be passaged through several generations and can be stored in liquid nitrogen and subsequently returned to culture. This work was supported by contract N01-CP75956 and grant R01-CA31733 from the National Cancer Institute. V. A. Varma is a recipient of an American Cancer Society fellowship; B. H. Dorman, a predoctoral fellowship from the Chemical Industry Institute of Toxicology; J. M. Siegfried, a training grant (CA09156) from the National Cancer Institute; and D. G. Kaufman, a Research Career Development Award (K04-CA-00431) from the National Cancer Institute.     

Decalcified egg shell membrane,a supporting substrate for electron microscopic cross-sections of monolayers of epithelial cell line MDCK

Summary Decalcified chicken egg shell membrane is a suitable physical substrate for the growth of monolayers of an epithelial line of tissue culture cells, MDCK. The shell membranes with their adherent sheets of cells are easily prepared for cross-section study of monolayer growth with electron microscopy. This study was supported by Research Grant P-442 from the American Cancer Society.     

Establishment and properties of a human choriocarcinoma cell line of ovarian origin

Summary A human nongestational choriocarcinoma cell line of ovarian origin (IMa) was established in vitro. This cell line had been subcultured serially more than 22 times over 18 months. Small polygonal cells with a prominent nucleus were dominant and a sparsity of cytoplasmic organelles was an ultrastructural characteristic of the IMa cells. The production and secretion of human chorionic gonadotropin and its subunits were identified by radioimmunoassay. The IMa cells were transplantable in the hamster cheek pouch and the histological diagnosis was choriocarcinoma. A newly established ovarian choriocarcinoma cell line can be considered useful for clarifying the biological differences between nongestational and gestational choriocarcinoma cells. This research was supported in part by a Grant-in-Aid for Cancer Research from both the Ministry of Health and Welfare and the Ministry of Education, Science, and Culture of Japan.     

Smooth muscle releases an epithelial cell scatter factor which binds to heparin

Summary We report that culture bovine calf aorta and human adult iliac artery smooth muscle cells release a soluble factor which causes spreading and separation of cells in normally tight, cohesive epithelial colonies, similar to the morphologic changes induced by the fibroblast-derived scatter factor (SF). Smooth muscle-derived SF was heat sensitive, trypsin labile, and nondialyzable, consistent with a protein (or proteins). Its effects on epithelium were not mimicked by a variety of proteolytic enzymes, growth factors, or hormones, and were not blocked by antiproteases or by antibodies to fibronectin and basic fibroblast growth factor. Epithelial cell proliferation was unaffected or only mildly stimulated by partially purified SF at concentrations that produced cell scattering. Both smooth muscle-and MRC5 human embryo fibroblast-derived SFs could be partially purified with similar elution patterns on a number of different chromatographic columns, including DEAE-agarose, heparin-sepharose, Bio-Rex 70, concanavalin A-sepharose, and MonoQ. SF from both sources bound tightly to heparin-sepharose, requiring 1.3 to 1.4M NaCl for elution. The morphologically obvious cell scattering effect was markedly inhibited by soluble heparin at concentrations down to 5 μg/ml, and this inhibition was prevented by protamine. These data suggest that vascular smooth muscle cells produce an epithelial cell scattering factor with properties similar to the fibroblast-produced factor, including a high affinity for heparin. Such factors are potentially important because they may represent a new class of proteins that primarily regulate cell mobility rather than growth and differentiation. Supported by American Cancer Society grant ACS IN-31-28-5, an Argail L. and Anna G. Hull Cancer Research Award, and grants-in-aid from the American Heart Association (#880981) and the American Lung Association of Connecticut. Dr. Goldberg was supported by the LIJ-Harvard Research Consortium and the Finkelstein Foundation.     

Epithelial outgrowth from suspension cultures of human prostatic tissue

Summary The primary objective of this study was to obtain pure cultures of prostatic epithelium. Encapsulation by epithelial cells and hypocellularity in stroma occurred when explants of prostatic tissue were maintained in suspension cultures. Twenty per cent fetal bovine serum incorporated into the medium provided optimal conditions for encapsulation and preservation of epithelial cell viability and architecture. Horse serum at the same concentration was less effective. When encapsulated explants were allowed to attach to the substrate, 10 and 20% horse serum favored growth of epithelial cells while fetal bovine serum also stimulated fibroblastic growth. Mechanisms for the induction of hypocellularity, encapsulation, squamous metaplasia and central necrosis in explants were studied. Relationship between the type and concentration of serum and the nature and extent of outgrowth are discussed. This work was supported in part by the American Cancer Society Grants DT-20 and IN-5N, U. S. Public Health Service Grant RR-05357, the Parke Davis Fund, and the John U. White Fund for Urological Research.     

Characterization of cells cultured from early lactation milks

Summary Two major types of cells can be cultured from early lactation human milks: a colony-forming epithelial cell and an adherent nondividing cell referred to as a foam cell The epithelial cells show a positive reaction with a specific antiserum reactive against membrane components of the milk fat globule, whereas the foam cells do not. The nondividing foam cells are phagocytic and can be killed by silica particles; they produce lysozyme, are resistant to trypsinization, and have Fc receptors. These properties, together with the lack of reaction with antiserum to the milk fat globule membrane, suggest that the foam cells are not terminally differential epithelial cells, but tissue macrophages. R. L. C. was supported by Grant No. Ca 19455 from the National Cancer Institute, a Yamagiawa-Yoshida Memorial International Cancer Study Grant, and the Imperial Cancer Research Fund. J. A. P. was supported by Grant No. CA 19455 from the National Cancer Institute.     

Attachment and multiplication,morphology and protein production of human fetal primary liver cells cultured in hormonally defined media

Summary We established for human fetal liver cells (cultured for 2 wk) in a hormonally defined medium, optimal conditions for attachment, multiplication, and preservation of epithelial morphology as well as production and secretion of serum proteins characteristic of fetal (α1-fetoprotein, AFP) and adult (albumin and hemopexin) life. Conditions were considered optimal when cell number, albumin, and hemopexin levels were maintained throughout the 2-wk culture period. However, the decrease in AFP concentration, which occurred after a few days of culture, could not be reversed. The culture system developed is a suitable model for studying regulatory mechanisms governing structure and function during differentiation and may prove useful for testing the effect of toxic agents during fetal development of the human liver. This work was supported by the Medical Research Council of Canada (Grant MA-7768), a grant from the “Fonds de la recherche en sante du Quebec” (F.R.S.Q.) and by “CAFIR” funds from the University of Montreal. David W. Barnes is supported by NIH-NCI-grants 40475 and 01226. MS-P was recipient of a scholarship from the Notre-Dame Hospital Foundation and from the Universite de Montreal.J-FT received a summer studentship from the FRSQ and from the Canadian Liver Foundation. Presented in preliminary form at the Tissue Culture Meeting in Houston, June 1984.     

The DNA-content (Feulgen) of nuclei during mitosis in a root tip of onion

Summary The microphotometric two-wavelength method is demonstrated to permit, even with only an improvised optical set-up, DNA-measurements which would not be possible, at any rate not with a comparable accuracy, with the conventional method.The data, in agreement with earlier results, show that the largest interphase nuclei in the root meristem have already completed their DNA-synthesis.The mean DNA-contents per set of chromatids at late interphase, prophase, and telophase, appeared to be virtually identical. A natural variation in DNA-content, if any, between individual nuclei at these stage would have a coefficient of variation of significantly less, and most likely much less, than 7.3%. The data from metaphase to middle anaphase are, for technical reasons, not yet conclusive. An indicated, moderate, excess of Feulgen-dye in these as compared with earlier and later stages may not be real.The results are fully compatible with a working hypothesis of strict constancy of the DNA-content per chromatid and of its precise doubling during DNA-synthesis (for necessary qualifications see the discussion). It is pointed out that apparently contradictory results by certain other authors do not, for methodological reasons, yet establish disproof of this hypothesis.Contribution from the Program in Cytology, Department of Botany, University of Wisconsin, Madison, supported in part by grants to Dr.C. Leonard Huskins from the Rockefeller Foundation, the American Cancer Society, and the Research Committee of the Graduate School with funds supplied by the Wisconsin Alumni Research Foundation.supported in part by the U. S. Public Health Service and the Wallace C. and Clara A. Abbott Memorial Fund.     

Establishment of a rat nasal epithelial tumor celline

Summary A new cell line designated NAS 2BL has been established from a rat nasal tumor induced by inhalation of the direct-acting carcinogen methylmethane sulfonate. The cells are epithelial in morphology, have a generation time of 34 h, require 10% fetal bovine serum for optimal growth, and exhibit keratinization at confluence. The karyotype is aneuploid, with several marker chromosomes, and the cells are transformed by the criterion of nude mouse turmorigenicity. This work was supported by grants CA36342, ES03563 and Center grants ES00260 and CA13343, from the National Institutes of Health; and Special Institutional grant 00009 from the American Cancer Society