Specificity of the cell-to-cell transmission of hormonal imprinting in cell cultures

Chang liver cells and Chinese hamster ovary (CHO) cells were imprinted either with insulin or with thyrotropin (TSH). Chang liver cells responded to insulin but not to TSH. As an effect of imprinting evoked by insulin administration the binding of insulin administered for the second time was enhanced. In the mixed culture of imprinted and intact cells the extent of the binding was similar to that seen in the cultures of the cells having received imprintatory treatment alone. CHO cells also responded to TSH, imprinting developed and was transmitted to the cells which were not in interaction with the hormone (intact cells). In CHO cells also insulin gave rise to imprinting for insulin, whereas TSH gave rise to moderate binding imprinting for insulin. On the other hand, insulin imprinting did not enhance the binding of TSH. The obtained results indicate that both the imprinting itself and the specificity of the transmission of imprinting depend on the characteristics of the cell-type in question. The extent of the transmission, however, is always proportional to the extent of imprinting.     

Cell-mediated transmission of hormonal imprinting to virgin mammalian cells in culture and failure of transmission with the nutrient medium

Chinese hamster ovary (CHO) cells and Chang liver cells which had already interacted with a hormone (gonadotropin, TSH, insulin) in culture, transmitted hormonal imprinting to virgin cells not previously involved in the interaction. The information associated with imprinting was not mediated by the nutrient medium, because the nutrient medium of the hormone-treated cells did not induce imprinting in virgin cells and even reduced rather than enhanced the hormone binding capacity thereof. Thus the transmission of information is in all probability associated with a direct cell-cell contact.     

Impact of combined hormonal pretreatment (insulin+TSH) on the imprinting of hormones administered in combination to Chinese hamster ovary cell culture.

Cultured Chinese hamster ovary (CHO) cells were treated (imprinted) with insulin and with thyrotropin (TSH) related to gonadotropins (FSH+LH). When one week later the treatment was repeated with one of the hormones, considerable differences could be observed in the binding capacity of the cells. In the hormone combination TSH was able to evoke persistent imprinting only to a markedly lesser degree than insulin, meanwhile the imprintatory effect of insulin was of greater extent even on the cell regarded to be unspecific for insulin. Hormone treatment of one hour duration--when investigated immediately after--did not extinct the binding capacity to TSH but enhanced that to insulin. With the deterioration of the conditions of culturing, the enhanced binding capacity disappeared.     

Influence of hormone concentration and time factor on TSH-FSH imprinting and overlap in CHO cells

Exposure of Chinese hamster ovarian cell cultures (cell line CHO) to TSH of FSH gave rise to hormonal imprinting. In earlier studies re-exposure after 48 h displayed a considerable increase in hormone binding. In the present experiments similar increase was demonstrated with an interval of five days. After 14 days, the increment was of lesser degree or even a decrease was noted in hormone-binding capacity. Although the CHO line originates from the target cells of gonadotropin, long-term positive imprinting was greater for TSH than for FSH, imprinting for FSH being negative rather than positive. The experimental results suggest that even very low concentrations (10(-13) mol) of hormone induce imprinting after an exposure as short as 60 min.     

Influence of receptor formation and receptor movement inhibitors on hormonal imprinting in cell culture

Hormonal imprinting takes place at the first interaction of the cell with the adequate hormone, and exerts a lasting influence on cellular binding capacity and functional response over many subsequent cell generations. Hormonal imprinting can also be induced in cell lines. In a Chinese hamster ovary (CHO K1) cell line, inhibitor of endocytosis and cellular protein synthesis inhibited hormone binding in themselves, and in cultures preexposed to TSH they inhibited imprinting by TSH in a dose-dependent manner. The protein synthesis inhibitor cycloheximide and the microfilament de-organizing agent cytochalasin-B inhibited imprinting by TSH to a greater degree than all other inhibitors tested, indicating that apart from cellular binding capacity, unimpaired cellular protein synthesis and microfilament activity are essential prerequisites of hormonal imprinting.     

Binding overlap and internalization of gonadotropin and thyrotropin in neonatal rat testicle and ovary cell cultures and the Chinese hamster ovary (CHO) cell line

Colloidal-gold-labeled gonadotropin and colloidal-gold-labeled thyrotropin were bound by Chinese hamster ovary (CHO) and primary neonatal rat ovary and testicle cell cultures in the same sites and in the same quantities. The conditions of internalization and the intracellular fate of the bound gold-labeled hormones were also similar in every respect. Pretreatment with either hormone imprinted the cells also for the related hormone, as judged from the increased binding and internalizing capacity of the pretreated cells for either hormone, and from identical patterns of post-binding receptor aggregation.     

Discrepancy in hormone binding and information transfer between sister cells of Tetrahymena clones

Tetrahymena cells treated with insulin in mass cultures were separated to single-cell clones or one of the "sister-cells" of dividing Tetrahymena (in single-cell culture) was treated with insulin. In both cases the FITC-insulin binding of sister-cells were compared. The insulin imprinting significantly increased the insulin binding of cells. There was also a significant difference between the imprinted and not imprinted sisters as well as between the not imprinted sisters. This demonstrates the existence of a difference (in hormone binding) between sister-cells and justifies that the information of the first hormone treatment (imprinting) is not equally divided between the sister-cells.     

Prolonged effect of an H1-receptor blocker antihistamine on the histamine content of white blood cells and mast cells

Hormonal imprinting usually takes place perinatally at the first encounter between the developing receptor and its target hormone, determining the future binding capacity of the receptor for life. Molecules similar to a hormone can cause faulty imprinting also with life-long consequences. Hormone production of the imprinted cell is also durably influenced. In cytogenic organs imprinting can also be provoked in adulthood. At present the effect of a single terfenadine treatment in adult rats on the histamine content of peritoneal cells (lymphocytes, mast cells and the monocyte-macrophage-granulocyte group), white blood cells (lymphocytes, granulocytes, monocytes) and thymic lymphocytes was studied 3 weeks after treatment to clarify the effect of prolonged treatment with an antihistamine in adulthood.The cells were studied by flow cytometric analysis. Peritoneal mast cells contained significantly more and thymic lymphocytes significantly less histamine than controls. In the other cells the differences were not significant. The results support earlier observations on the effect of antihistamines on mast cell histamine release (inhibition) and call attention to the fact that this effect is durable (hormonal imprinting provoked in adults).     

Permanence of the cell-to-cell transmission of insulin induced hormonal imprinting

When insulin-treated (imprinted) Chang liver cell cultures were mixed with cultures which did not receive insulin treatment the information of imprinting was transmitted to the cultures which were not in direct contact with insulin. The ability of the cells to transmit imprinting was long lasting and could be detected even after four weeks, when it was nearly of the same degree as at the first measurement. Difference was found between the binding capacity of the receptors of the plasma membrane and those of the nuclear membrane.     

Insulin pretreatment (imprinting) produces elevated capacity in the insulin binding of Tetrahymena. Different binding by the cilia of the body and oral field

Gold-labeled insulin is bound first of all to the cilia of the oral field of Tetrahymena. A primary treatment (hormonal imprinting) with insulin increases the binding capacity even after 24h and makes it more sensitive for appearance a week later, within a minute of giving insulin-gold. The food vacuoles contain insulin-gold in pretreated cells or without pretreatment as well, though in imprinted situations the label can be found in pinocytotic vesicles at the bases of cilia in the oral field. Altogether, a functional difference can be observed between the cilia of the oral and non-oral surfaces of Tetrahymena and hormonal imprinting has a specifying effect on the binding of labeled hormone.     

Influence of hormone treatment applied or begun in different phases of the cell cycle on hormonal imprinting in Tetrahymena

Primary exposure to a hormone (hormonal imprinting) alters--in the case of the Tetrahymena increases--cellular response to re-exposure(s) to the same hormone. The intensity of hormonal imprinting depends on the phase of the cell cycle in which the primary exposure has taken place. The effect of imprinting was greater on the cells exposed to the hormone in phase G1 than on those exposed in phase S or G2. The response pattern of the progeny generations corresponded to that of the primarily exposed (imprinted) ancestor cell, irrespective of their own pre-exposure in phase G1, G2 or S of their cycle.     

Hormonal imprinting in cell culture II. Evidence of hormonal imprinting and thyrotropin (TSH) -gonadotropin (FSH) overlap in a Chinese hamster ovary (CHO) cell line

Reexposure of cultures of the Chinese hamster ovarian cell line CHO K1 to FITC-labeled hormone 48 h after the first 24-h exposure to FSH or TSH showed that hormonal imprinting, accounting for a greater binding capacity on reexposure, also took place in in vitro conditions. TSH amplified the receptors of FSH to a greater degree than FSH itself, although the reverse effect failed to happen. TSH was able to bind the ovarian cells at first exposure, and to amplify the receptors for itself and--remarkably--to a considerably greater degree for FSH, exactly as observed earlier in in vivo systems.     

Surface imprinted beads for the recognition of human serum albumin

The synthesis of poly-aminophenylboronic acid (ABPA) imprinted beads for the recognition of the protein human serum albumin (HSA) is reported. In order to create homogeneous recognition sites, covalent immobilisation of the template HSA was exploited. The resulting imprinted beads were selective for HSA. The indirect imprinting factor (IF) calculated from supernatant was 1.6 and the direct IF, evaluated from the protein recovered from the beads, was 1.9. The binding capacity was 1.4 mg/g, which is comparable to commercially available affinity materials. The specificity of the HSA recognition was evaluated with competitive experiments, indicating a molar ratio 4.5/1 of competitor was necessary to displace half of the bound HSA. The recognition and binding of the imprinted beads was also tested with a complex sample, human serum and targeted removal of HSA without a loss of the other protein components was demonstrated. The easy preparation protocol of derivatised beads and a good protein recognition properties make the approach an attractive solution to analytical and bio-analytical problems in the field of biotechnology.     

Design and synthesis of molecularly imprinted polypyrrole based on nanoreactor SBA-15 for recognition of ascorbic acid

Molecular imprinting is an attractive technique for preparing mimics of natural and biological receptors. Nevertheless, molecular imprinting for aqueous systems remains a challenge due to the hydrogen bonding between templates and functional monomers destroyed in the bulk water. The hydrogen bonding between templates and monomers are the most crucial factor governing recognition, particularly in non-covalent molecularly imprinted polymers. Using mesoporous materials for molecular imprinting is an effective approach to overcome this barrier and to remove the limitations of the traditional molecularly imprinted polymers which include incomplete template removal, small binding capacity, slow mass transfer, and irregular materials shape. Here, SBA-15 was used as a mesoporous silica material for synthesis of molecularly imprinted polypyrrole. The pyrrole monomers and template molecules were immobilized onto the SBA-15 hexagonal channels, and then polymerization occurred. The resulting nanocomposites were characterized by Fourier transform infrared (FT-IR) analysis, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) methods. In batch rebinding tests, the imprinted nanocomposites reached saturated adsorption within 100min and exhibited significant specific recognition toward the ascorbic acid (AA) with high adsorption capacity (83.7mgg(-1)). To further illustrate the recognition property of the imprinted nanocomposites, binary competitive and non-competitive adsorption experiments were performed with ascorbic acid, dopamine, paracetamol and epinephrine. The imprinting factors for these compounds in non-competitive adsorption experiments were 3.2, 1.5, 1.4 and 1.3, respectively. The results showed that the imprinted nanocomposites exhibited significant adsorption selectivity for the ascorbic acid against the related compounds.     

A loss of insulin-like growth factor-2 imprinting is modulated by CCCTC-binding factor down-regulation at senescence in human epithelial cells

The imprinted insulin-like growth factor-2 (IGF2) gene is an auto/paracrine growth factor expressed only from the paternal allele in adult tissues. In tissues susceptible to aging-related cancers, including the prostate, a relaxation of IGF2 imprinting is found, suggesting a permissive role for epigenetic alterations in cancer development. To determine whether IGF2 imprinting is altered in cellular aging and senescence, human prostate epithelial and urothelial cells were passaged serially in culture to senescence. Allelic analyses using an IGF2 polymorphism demonstrated a complete conversion of the IGF2 imprint status from monoallelic to biallelic, in which the development of senescence was associated with a 10-fold increase in IGF2 expression. As a mechanism, a 2-fold decrease in the binding of the enhancer-blocking element CCCTC-binding factor (CTCF) within the intergenic IGF2-H19 region was found to underlie this switch to biallelic IGF2 expression in senescent cells. This decrease in CTCF binding was associated with reduced CTCF expression in senescent cells. No de novo increases in methylation at the IGF2 CTCF binding site were seen. The forced down-regulation of CTCF expression using small interfering RNA in imprinted prostate cell lines resulted in an increase in IGF2 expression and a relaxation of imprinting. Our data suggest a novel mechanism for IGF2 imprinting regulation, that is, the reduction of CTCF expression in the control of IGF2 imprinting. We also demonstrate that altered imprinting patterns contribute to changes in gene expression in aging cells.     

Studies into hormonal imprinting in intact and transformed 3T3 fibroblast cell lines

The cells of the NIH 3T3 fibroblast line responded to primary interaction with insulin by a positive imprinting, i.e. by an increased binding capacity for the hormone on re-exposure. Positive imprinting, although to a lesser degree, was also induced by thyrotropin. However, oncogenic transformation by polyoma virus oncogens resulted in decreased imprinting in both the middle-T-antigen (MT3) and small-T-antigen-expressing (N4) cells.     

Impact of serum concentration of the medium and fasting on the imprintability of the insulin receptors of Chang liver cells.

When the cells of the Chang cell line came into interaction with a hormone (insulin) an imprinting-like phenomenon took place. The binding capacity of the receptors strengthened and this feature was transmitted to the descendant generations. The quality of the nutrient medium influenced the development of imprinting, when the cells were maintained in a medium containing 2% serum it was more difficult to evoke imprinting than in case the cells were kept in a medium containing 10% serum. If the cells were cultured kept in Tyrode (physiological) solution for 24 hours the possibility to evoke imprinting was lost. Difference could be observed between the behaviour of receptors in nuclear membrane and that of receptors in the plasma membrane; i.e. changes were more dynamic in the plasma membrane.     

Impact of 5-azacytidine on insulin binding and insulin-induced receptor formation in tetrahymena.

The unicellular Tetrahymena is able to bind the vertebrate hormone insulin, and the binding sites presented by it become amplified under hormonal influence. The increased binding capacity for insulin reappears in many offspring generations. 5-azacytidine inhibits insulin binding and the insulin-induced formation of binding sites as well in the cell generation directly involved in interaction, but enhances insulin binding in the daughter cell generations. The nutrient medium of the cells whose binding capacity was enhanced by azacytidine treatment transmitted the information accounting for increased binding to "virgin" cells not previously treated with azacytidine.     

Molecularly imprinted thin film self-assembled on piezoelectric quartz crystal surface by the sol-gel process for protein recognition

A novel method of combining sol-gel and self-assembly technology to prepare a human serum albumin (HSA)-imprinted film on the surface of piezoelectric quartz crystal (PQC) Au-electrode modified with thioglycolic acid was described in this paper. The imprinting process was characterized by using the piezoelectric quartz crystal impedance (PQCI) technique and electrochemical impedance technique. Scanning electron microscope (SEM) was employed to characterize the surface morphology of the resultant imprinted film. The piezoelectric technique and electrochemical impedance technique were also employed to investigate the binding performance of the sol-gel-imprinted film with the template protein. The results showed that the imprinted PQC film can give selective recognition to the template protein. The effects of salts and solvents on the binding capacity of the imprinted film with protein were discussed in detail. Other influencing factors (temperature and pH) have also been investigated. This self-assembly sol-gel imprinting technique was proved to be an alternative method for the preparation of biomacromolecule-imprinted thin film.     

Activation of paternally expressed imprinted genes in newly derived germline-competent mouse parthenogenetic embryonic stem cell lines

Parthenogenetic embryonic stem （pES） cells provide a valuable in vitro model system for studying the molecular mechanisms that underlie genomic imprinting. However, the pluripotency of pES cells and the expression profiles of paternally expressed imprinted genes have not been fully explored. In this study, three mouse pES cell lines were established and the differentiation potential of these cells in extended culture was evaluated. The undifferentiated cells had a normal karyotype and homozygous genome, and expressed ES-cell-specific molecular markers. The cells remained undifferentiated after more than 50 passages and exhibited pluripotent differentiation capacity. All three lines of the established ES cells produced teratomas; two lines of ES cells produced chimeras and germline transmission. Furthermore, activation of the paternally expressed imprinted genes Snrpn, U2afl-rsl, Peg3, Impact, Zfp127, Dlkl and Mest in these cells was detected. Some paternally expressed imprinted genes were found to be expressed in the blastocyst stage of parthenogenetically activated embryos in vitro and their expression level increased with extended pES cell culture. Furthermore, our data show that the activation of these paternally expressed imprinted genes in pES cells was associated with a change in the methylation of the related differentially methylated regions. These findings provide direct evidence for the pluripotency of pES cells and demonstrate the association between the DNA methylation pattern and the activa- tion of paternally expressed imprinted genes in pES cells. Thus, the established ES cell lines provide a valuable model for studying epigenetic regulation in mammalian development.