Estrogen-dependent DNA synthesis and parenchymal cell proliferation in the liver of adult male Xenopus frogs

Estradiol-17 beta treatment of adult male Xenopus laevis induces liver parenchymal cells to synthesize DNA and proliferate. DNA synthesis begins 3 to 4 days after estrogen treatment and continues for approximately 10 days. Over this 2-week period, the total number of liver parenchymal cells increases fourfold, the wet weight of the liver remains constant, and there is a 50% reduction in cell volume. The elevated number of cells persists for several months and then returns to the control value. The extent of proliferation is hormone dose dependent. Pulse-chase experiments demonstrate that as a result of hormone treatment a minority of the parenchymal cells in the initial population enter the cell cycle, and via repeated divisions become the majority (79%) of the population by Day 14. The implications of this phenomenon for estrogen-induced liver cell differentiation and vitellogenin gene function are discussed.     

Thyroid hormones are corequisites for estradiol-17β in vitro induction of Xenopus vitellogenin synthesis and secretion

Estradiol-17β administered to male frogs induces liver synthesis and secretion of vitellogenin, the precursor protein of the major egg yolk proteins. Estradiol-17β alone failed to induce this protein in cultures of liver tissue maintained for 1–2 weeks prior to addition of the hormone. If a “complex” defined culture medium, such as Coon's modified Ham's F12 medium, is used, efficient primary and secondary induction of vitellogenin synthesis and secretion occurs in the presence of estradiol-17β, triiodothyronine, and dexamethasone. Using Coon's medium we investigated the role of both triiodothyronine and dexamethasone as corequisites of estradiol-17β induction of secreted vitellogenin. Control cultures given no hormones showed a gradual decrease in the level of secreted albumin and fibrinogen. Addition of dexamethasone, alone, induced increased synthesis of secreted albumin and fibrinogen as well as other proteins. Cultures given thyroid hormones, alone, showed an increased level of secreted albumin and fibrinogen at early time points in the culture period. Thus, at early times thyroid hormones appear to enhance the activity of endogenous glucocorticoids. Independent of their interaction with glucocorticoids, thyroid hormones also enhance the activity of estrogens. Long-term cultures given estradiol-17β, alone, failed to synthesize and secrete vitellogenin. In contrast, cultures given the estrogen together with thyroid hormones showed vitellogenin synthesis. These results imply that similar interactions of several hormones occur in vivo in adult animals treated with estrogens. In the accompanying paper the interaction of dexamethasone with estradiol-17β and triiodothyronine is described (L. J. Wangh, 1982, Develop. Biol.89, 294–298).     

Cell growth and cell proliferation may be dissociated in the mouse uterine luminal epithelium treated with female sex steroids

The mouse uterine epithelium under various hormonal regimes is a good system to identify biochemical events associated with cell growth, DNA synthesis and cell division. This is because estradiol-17β stimulates the cells to undergo a synchronized wave of DNA synthesis and cell division. Estriol, on the other hand, also stimulates DNA synthesis but because of the rapid loss of this hormone from the tissue some of the cells abort, giving a constant epithelial cell number. Three days of progesterone pretreatment, however, completely suppresses the estradiol-17β-induced wave of DNA synthesis and cell proliferation.Using these hormonal treatments we have shown that both estradiol-17β and estriol stimulate protein and rRNA synthesis with the concomitant increase of protein and rRNA per mg of DNA. These macromolecules accumulated in direct proportion to the fraction of cell committed to DNA synthesis. Estriol, however, did not sustain the growth responses and at the peak of DNA synthesis both rRNA and protein synthesis had returned to control levels. Progesterone pretreatment, despite inhibiting the proliferative response, failed to inhibit any of the estradiol-17β-induced increases in protein and rRNA synthesis. Indeed 12 h after estradiol-17β injection the cells had identical protein and rRNA contents, regardless of whether they had been exposed to progesterone or not.The present data therefore suggests that in the uterine epithelium cell growth as defined by protein and rRNA accumulation and DNA synthesis represents two independently regulated pathways.     

Glycogen synthesis in serum-free cultured hepatocytes in response to insulin and dexamethasone

Liver parenchymal cells cultured in serum-free medium may retain their ability to synthesize glycogen in response to insulin. Specific hormone requirements are needed by hepatocytes to retain the biochemical pattern of mature cells. Insulin supplementation of culture medium seems to be essential to maintain the glycogen synthesis rate of cultured hepatocytes. The continuous presence of dexamethasone amplified the insulin-induced glycogen synthesis. Cytophotometric analysis showed differences in the way that individual cells accumulate glycogen in response to insulin stimulus, which indicates that liver parenchymal cells in culture are functionally heterogeneous.     

Parenchymal cells purified from Xenopus liver and maintained in primary culture synthesize vitellogenin in response to estradiol-17 beta and serum albumin in response to dexamethasone.

Xenopus liver was disaggregated by perfusion with collagenase. The released cells were separated according to their equilibrium densities on gradients of Metrizamide. Highly purified populations of parenchymal cells, sinusoidal cells, and melanocytes were obtained. All the parenchymal cells in an animal, before, during, and after hormone stimulation, are contained within a single density peak and subpopulations are not observed. Normal female parenchymal cells are less dense than normal male cells and estrogen stimulation causes a decrease in the peak density of the parenchymal cell population. Electron microscopic examination revealed the appearance and disappearance of lipid vacuoles in parenchymal cells following estrogen treatment. We hypothesize that the changing lipid content of these cells accounts for the shift in cell density. Immunofluorescent staining revealed that all liver parenchymal cells contain either vitellogenin or serum albumin, depending upon the prior treatment with hormones in vivo. Primary cultures of purified parenchymal and sinusoidal cells were established and were found to secrete different sets of proteins. The addition of estradiol-17β to cultures of parenchymal cells from either male or female frogs induces the synthesis and secretion of vitellogenin and two other polypeptides. Furthermore, the synthesis and secretion of polypeptides normally produced by parenchymal cells are curtailed as a result of estrogen treatment. Addition of glucocorticoids to a similar population of cells produces an increase in the synthesis and secretion of polypeptides, one of which is serum albumin. These results demonstrate that every parenchymal cell is responsive to two different classes of steroid hormones, estrogens and glucocorticoids, and in response to these hormones the same cells can synthesize and secrete alternate sets of polypeptides.     

Parenchymal cells purified from Xenopus liver and maintained in primary culture synthesize vitellogenin in response to estradiol-17β and serum albumin in response to dexamethasone

Xenopus liver was disaggregated by perfusion with collagenase. The released cells were separated according to their equilibrium densities on gradients of Metrizamide. Highly purified populations of parenchymal cells, sinusoidal cells, and melanocytes were obtained. All the parenchymal cells in an animal, before, during, and after hormone stimulation, are contained within a single density peak and subpopulations are not observed. Normal female parenchymal cells are less dense than normal male cells and estrogen stimulation causes a decrease in the peak density of the parenchymal cell population. Electron microscopic examination revealed the appearance and disappearance of lipid vacuoles in parenchymal cells following estrogen treatment. We hypothesize that the changing lipid content of these cells accounts for the shift in cell density. Immunofluorescent staining revealed that all liver parenchymal cells contain either vitellogenin or serum albumin, depending upon the prior treatment with hormones in vivo. Primary cultures of purified parenchymal and sinusoidal cells were established and were found to secrete different sets of proteins. The addition of estradiol-17β to cultures of parenchymal cells from either male or female frogs induces the synthesis and secretion of vitellogenin and two other polypeptides. Furthermore, the synthesis and secretion of polypeptides normally produced by parenchymal cells are curtailed as a result of estrogen treatment. Addition of glucocorticoids to a similar population of cells produces an increase in the synthesis and secretion of polypeptides, one of which is serum albumin. These results demonstrate that every parenchymal cell is responsive to two different classes of steroid hormones, estrogens and glucocorticoids, and in response to these hormones the same cells can synthesize and secrete alternate sets of polypeptides.     

Effects of interaction between estradiol-17 beta and progesterone on the proliferation of cloned breast tumor cells (MCF-7 and T47D)

We have previously reported that the proliferation of cloned MCF-7 and T47D human mammary tumor cells can be inhibited by increasing concentrations of charcoal-dextran stripped female human serum (CDFHS). The maximal proliferation rate was restored by the addition of 3 X 10(-11) M estradiol-17 beta to the culture media. These observations suggest that the proliferation of T47D and MCF-7 cells is regulated by a blood-borne inhibitor whose effects are neutralized by estrogens. In the present report we explore the possibility that progesterone alters the estrogenic response. MCF-7 cells were grown in DME containing 2-40% CDFHS. Progesterone, at 3 X 10(-7) M to 3 X 10(-12) M, had no effect on the yield of MCF-7 or T47D cells that were cultured in the presence or absence of estradiol-17 beta.     

Estrogen receptors and growth response in cultured human periodontal ligament cells

The periodontal ligament (PDL) is a connective tissue involved in the remodeling process associated with tooth development and positioning. PDL cells grown in culture were analyzed for the capacity to specifically bind steroid hormones and for growth response to estradiol-17 beta. Using [3H]estradiol-17 beta as the ligand, PDL cells in first passage cultures exhibited a specific estrogen binding capacity of 881 fmol/mg cell protein. With [3H]dexamethasone as a ligand, the binding capacity of the glucocorticoid receptor was 143 fmol/mg protein. With [3H]R5020 as a ligand, the progestin receptor exhibited a binding capacity of 5 pmol/mg protein. Scatchard analysis of estradiol binding at 37 degrees revealed a dissociation constant of 2.7 X 10(-9) M, representative of the estrogen receptor. The addition of estradiol-17 beta at concentrations of 10(-9) and 10(-8) M to culture media induced a dose-dependent decrease in growth (DNA content) to 62% and 38% control values, respectively. The addition of the antiestrogens tamoxifen and 4-hydroxytamoxifen at concentrations of 10(-7) and 10(-6) M similarly depressed cell growth. These results show that PDL cells contain high affinity receptors for several steroid hormones and further that these cells are targets for the action of estrogens.     

Glycogen synthesis in serum-free cultured hepatocytes in response to insulin and dexamethasone

Summary Liver parenchymal cells cultured in serum-free medium may retain their ability to synthesize glycogen in response to insulin. Specific hormone requirements are needed by hepatocytesto retain the biochemical pattern of mature cells. Insulin supplementation of culture medium seems to be essential to maintain the glycogen synthesis rate of cultured hepatocytes. The continuous presence of dexamethasone amplified the insulin-induced glucogen synthesis. Cytophotometric analysis showed differences in the way that individual cells accumulate glycogen in response to insulin stimulus, which indicates that liver parenchymal cells in culture are functionally heterogeneous. The financial support for this work was from the Fondo de Investigationes Sanitarias de la Seguridad Social, grants 41/82 and 48/82.     

The Role of Hepatotrophic Factors in Liver Regeneration — A Brief Review Including a Preliminary Report of the In Vitro Effects of Hepatic Regenerative Stimulator Substance (SS)

Details are presented of a technique for maintaining adult liver parenchymal cells in culture in a nonproliferating state. Hepatic regenerative stimulator substance (SS), prepared from weanling rat liver, will stimulate tritiated thymidine incorporation into liver DNA when injected into another rat or mouse. Preliminary experiments are presented which show that SS stimulates non-proliferating adult hepatocytes to incorporate increased amounts of ³HT dr into DNA when SS is added to the culture medium. The response is dose dependent and a lag period of about 12 hours exists between addition of SS to the cultures and the stimulation of DNA synthesis. Preliminary screening of several cell lines in culture reveals that only liver related cell lines (hepatomas) respond to SS. A brief review of other hepatotrophic factors is also included.     

In vitro induction of luteinizing hormone synthesis by estrogen in organ-cultured pituitary glands of the Japanese eel,Anguilla japonica

An organ culture method for pituitary glands isolated from immature Japanese eels (Anguilla japonica) was developed. This method could conserve the histological features of the pituitary glands for at least 21 days. The ability to synthesize gonadotropic hormone (GTH) in cultured eel pituitary glands was examined by detecting luteinizing hormone (LH) beta protein immunohistochemically. In a basal medium (Leibovitz L-15), LH beta-immunoreactive cells were very scarce, but after addition of estradiol-17beta (E2) a large number of immunoreactive cells appeared, particularly in the proximal pars distalis. The stimulatory effects of E2 on LH beta synthesis were dose (1-100 ng/ml)- and time (1.5-7 days)-dependent. Thus, in contrast with previous reports of the lack of a direct effect of E2 on GTH synthesis in primary cultured eel pituitary cells, the present results clearly indicate that E2 can stimulate GTH synthesis in immature eel pituitary glands. This organ culture method is useful to examine the actions of steroids and also other endocrine factors on the eel pituitary gland.     

Neurokinin A induces c-fos, c-jun, and c-myc expression in L6 rat myoblasts

Neurokinin A (NKA), a neuropeptide belonging to the tachykinin family, induced c-fos proto-oncogene mRNA expression in serum-deprived L6J1 rat skeletal myoblasts in vitro. The marked increase reached maximal levels after 15 to 30 min. In contrast to this, c-jun and c-myc proto-oncogene expression were only slightly induced, with peak levels after 30 min. NKA did not stimulate DNA synthesis or cell proliferation in serum-deprived L6J1 myoblasts. We demonstrate a relationship between NKA treatment and induction of c-fos, c-jun and c-myc mRNA expression in serum-deprived L6J1 rat myoblasts. The results on DNA synthesis and cell proliferation indicate that the induced proto-oncogene expression alone is not enough to induce a cellular response to NKA. Possible mechanisms of action are discussed.     

The estrogenic responses to clomiphene in the different cell types of the rat uterus: morphometrical evaluation

The present study was designed to investigate the dose-response of clomiphene on several estrogenic responses in the immature rat uterus and to compare it to available data on estradiol-17 beta. A dissociation was demonstrated among the different estrogenic responses induced by clomiphene. Very high doses of clomiphene were needed to induce the 6-h uterine eosinophilia and deep endometrial edema, and maximal response levels were not reached at any dose studied. On the contrary, many genomic responses were induced with much lower doses of clomiphene, and maximal response levels were reached with at least the two highest doses of clomiphene. This dissociation is in agreement with the existence of separate groups of responses that are mediated by multiple and independent mechanisms of estrogen action involving different kind of receptors. Luminal epithelial, glandular epithelial, and myometrial hypertrophies were also found to differ with regard to the dose needed to induce this response in each cell type. The dissociation between genomic responses of the different uterine cell types supports the hypothesis of different estrogen receptors for each kind of cell. Clomiphene induces mitoses in the different cell types, but the proportion of mitoses in the cell types was different from that described for estradiol. It is suggested that these differences are also due to differences between receptors involved in cell proliferation.     

THE KINETICS OF CELLULAR PROLIFERATION IN REGENERATING LIVER

The study concerns the kinetics of cellular proliferation in the different cell populations of the normal and regenerating rat liver. A detailed analysis is presented, which includes techniques of in vivo labeling of DNA with tritiated thymidine and high-resolution radioautography, of the temporal and spatial patterns of DNA synthesis and cell division in the parenchymal cells, littoral cells, bile duct epithelium, and other cellular components in the liver during the first 64 hr of regeneration after partial hepatectomy. The analysis of cell population kinetics indicates that (a) the rate of entry of parenchymal cells into synthesis, after an initial burst of proliferative activity, was an orderly progression at 3–4%/hr; (b) most cells divided once and a few twice, a large proportion of the cell deficit being replaced by 72 hr after the onset of proliferation; (c) T_s was ~8.0 hr; T_gg2+m/2, 3.0 hr; and M, ~1.0 hr. Littoral cell proliferation began about 24 hr after the onset of parenchymal cell proliferation; the rate of entry of littoral cells into synthesis was greater than 4%/hr. Interlobular bile duct cell proliferation lagged well behind the parenchymal and littoral cell populations both in time and extent of proliferation.     

Major loss of the 28-kD protein of gap junction in proliferating hepatocytes

There is a reduction in the 28-kD gap junction protein detectable by immunofluorescence in livers of partially hepatectomized rats and in cultured hepatocytes stimulated to proliferate. By the coordinate use of antibodies directed to the hepatic junction protein (HJP28) and the use of a monoclonal antibody that recognizes bromodeoxyuridine (BrdU) incorporated into DNA, we have been able to study the relationship between detectable gap junction protein and cell division. Hepatocytes that label with BrdU in the regenerating liver and in cell culture show a significant reduction of HJP28. Cells that do not synthesize DNA, on the other hand, show normal levels and distribution of immunoreactive gap junction protein. We postulate that the quantitative changes in gap junction expression might play an important role in the control of proliferation in the liver.     

Glycosaminoglycan synthesis by liver parenchymal cell clones in culture and its change with transformation

Albumin-producing rat liver parenchymal cell clones (BB and BC) and their subclones in the confluent culture synthesized heparan sulfate as the major component and dermatan sulfate, chondroitin sulfate and hyaluronic acid as the minor ones. Their relative contents were similar to those present in the rat liver.Analyses of glycosaminoglycans synthesized by subclone cells (BB1S) at various cell densities, cell growth phases and passage levels have shown that relative content of heparan sulfate remained constant, suggesting that the epithelial cell possesses a stable heparan sulfate-producing capacity. On the other hand, the level of hyaluronic acid production was high at low cell density, though it remained constant during cell proliferation.Chemically transformed rat liver parenchymal cells (M) produced relatively higher amount of chondrotin sulfate than non-transformed cells did, as observed with 4-nitroquinoline-1-oxide-transformed 3T3 cells, compared to 3T3 714 cells.The results obtained in this study strongly suggest that the liver parenchymal cells synthesize a major part of the glycosaminoglycans of the liver and chondroitin sulfate production is closely related to cellular proliferations.     

石竹细胞悬浮培养研究

石竹细胞继代周期为 7d时 ,悬浮细胞培养系生长最快 ,生长率最高 ,而且培养物中胚性细胞较多 ,并能保持较快的分裂和生长 ,能促进已形成的大细胞团的生长和分化。转代时接种物与新鲜培养基的体积比以1∶2较好 ,悬浮系细胞生长最快 ,生长率最高 ,以 1∶2和 1∶3的高倍稀释接种有利于胚性细胞的形成及产生小的胚性细胞团 ,对悬浮系添加椰乳和水解乳蛋白的混合物 ,可较大幅度地提高悬浮细胞系的生长速率 ,单独添加上述两种物质的效果均不如二者的综合效应好。在 6种不同激素组合中 ,配方 2 (2 ,4 D 1 .5mg/L +NAA0 .5mg/L +6 BA 0 .5mg/L)最好 ,生长率最高。配方 5 (2 ,4 D 1 .5mg/L +NAA 0 .5mg/L +6 BA 1 .0mg/L)其次 ;配方 1 (2 ,4 D 1 .0mg/L +NAA 0 .5mg/L +6 BA 0 .5mg/L)次之。     

Genotoxic effects of estradiol-17beta on human lymphocyte chromosomes

The cytogenetic effect of a hormonal steroid, estradiol-17beta, was assessed in peripheral blood human lymphocyte culture. Sister chromatid exchanges (SCE) and chromosome aberrations (CA) were scored as genetic end points. Significant induction of CA was observed at 25 microg/ml and 50 microg/ml concentrations of estradiol-17beta in the absence of microsomal activation. The drug was effective in all treatments in the presence of rat liver S(9) microsomal fraction (S(9) mix) and exhibited increased frequency of chromosomal aberrations. The drug was effective in increasing the SCE frequency which was found to be maximum at the dose of 50 microg/ml concentration (i.e., 4.34+/-1.22) both with and without metabolic activation. It was found that estradiol-17beta itself and possibly its metabolites are potent mutagens beyond a particular dose in human lymphocytes.     

Histopathology and cell culture characteristics of liver cells from grc − and grc + rats given diethylnitrosamine

The histopathological response and cell culture characteristics of liver cells from the R16 (grc ^–) strain of rats, which carries an MHC-linked deletion, were examined one week after a single intraperitoneal injection of 200 mg/ kg body weight diethylnitrosamine (DEN) and were compared with the response of liver cells from wild type (grc+) rats. The DEN exposure induced hydropicl vacuolar changes in the parenchymal cells and a limited proliferation of oval cells in the periportal areas of the livers of both grc+ and grc rats. Primary culture of collagenase-digested livers consisted of parenchymal, bile ductular and oval-related cells as determined by cell-specific immunohistochemistry. Subpassaged cells from grc+ rats exhibited oval cell ultrastructural morphology, inducible histochemical staining for gammaglutamyl transpeptidase (GGT), and DEN-associated onset of anchorage-independent growth. Primary cultures of liver cells from R16 rats consistently failed to form cell strains upon subpassage.Abbreviations DEN diethylnitrosamine - grc growth and reproduction complex - GGT gamma-glutamyl transpeptidase - MHC major histocompatibility complex     

The necessity for T cell help for human tonsil B cell responses to pokeweed mitogens: induction of DNA synthesis, immunoglobulin, and specific antibody production with a T cell helper factor produced with pokeweed mitogen.

Human B lymphocytes obtained from tonsils do not proliferate when stimulated with pokeweed mitogen. A soluble factor produced from T cells cultured with pokeweed mitogen stimulates B cells to synthesize DNA and differentiate into immunoglobulin producing cells. This PWM produced supernatant induced a PFC response to SRBC. The T cell supernatant activity is produced within 12 hr of stimulation in the presence of serum and without a requirement for T cell division. Optimal stimulation of B cells occurred at 7 to 9 days of culture. This helper factor activity eluted postalbumin from a column of Sephadex G-200. Insolubilized pokeweed mitogen was not mitogenic for B cells. The continuous presence of the lectin in culture was not required for B cell proliferation or for immunoglobulin synthesis.