An attempt at transmission of hormonal imprinting between foreign cell lines

Hormonal imprinting is transmitted from imprinted to virgin cells of the same cell line. No transmission of imprinting occurred between Chinese hamster ovary (CHO) and human Chang liver cells, and the presence of the latter reduced rather than enhanced the thyrotropic hormone (TSH) binding capacity of the CHO cells. While hormone binding capacity was relatively homogeneous in the control and the mixed cell cultures, it was not homogeneous in the homologous imprinted plus virgin cell population, indicating a continuous transmission of imprinting from the former to the latter.     

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.     

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.     

Influence of interference with the cyclic AMP-adenylcyclase-phosphodiesterase system on TSH-induced hormonal imprinting in the Tetrahymena

Primary interaction of TSH with the unicellular Tetrahymena accounted for an increase in TSH binding capacity on reexposure, i.e. for a regular hormonal imprinting. TSH in itself did not give rise to a faulty imprinting (for insulin). Combination of TSH with dibutyryl cAMP reduced the intensity of imprinting, whereas theophylline or lithium ions not only reduced the efficacy of normal imprinting, but also gave rise to faulty imprinting (for insulin instead of TSH).     

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.     

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.     

Studies into hormonal imprinting in a Tetrahymena thermophila mutant incapable of lysosomal enzyme secretion

Reexposure to insulin after primary interaction (hormonal imprinting) was followed by a binding increase in T. pyriformis and by a binding decrease in T. thermophila. The sec. mutant, MS-1 strain of T. thermophila, which is unable of lysosomal enzyme secretion, also showed a binding increase on a second exposure to insulin, from which it follows that alteration of the enzyme secretion, or other factors associated with mutation, accounted for reversion of the trend of imprinting. Thyrotropic hormone (TSH) also gave rise to a negative imprinting in T. thermophila, but did not alter the binding relations of the MS-1 mutant strain.     

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.     

Effect of inhibitors and activators of tyrosine kinase on insulin imprinting in Tetrahymena.

Primary exposure of Tetrahymena cells to insulin gave rise to hormonal (insulin) imprinting in the offspring generations, as judged from the increase in binding upon reexposure to insulin. Vanadate mimicked the action of insulin, inasmuch as it also induced imprinting for insulin, whereas the other tyrosine kinase activator tested, namely H2O2, had no such effect. However, combined treatment with vanadate+H2O2 + insulin induced a more pronounced imprinting for insulin than either insulin or vanadate on their own. The tyrosine kinase inhibitor genistein, a plant flavonoid, did not change the value for insulin binding significantly relative to the control immediately after exposure, but increased it slightly in the offspring generations after 24 h at high dilution. Upon combination with insulin, 10(-4)M genistein inhibited imprinting by insulin. These experimental observations suggest that there may be a key role for tyrosine kinase activity in the mechanism (development) of imprinting.     

Interrelationships between the imprinting potential and biological activity of insulin derivatives: studies on Tetrahymena and Chang liver cells

Insulin dimers deprived of biological activity by linking with suberic acid symmetrically in position B29 or B1 were not able to induce imprinting. Lack of N-terminal phenylalanine or even of five C-terminal amino acids did not interfere with imprinting, regardless of whether or not it was associated with an activity loss. It appears that while hormonal imprinting is closely associated with the hormone's ability to bind to the receptor, it may be related as well as unrelated to the hormone's biological activity. The imprinted Tetrahymena and Chang cells bound the insulin and its derivatives in a similar manner.     

The role of Ca2+ in hormonal imprinting of the Tetrahymena

It is known from model experiments on Tetrahymena that primary exposure to a hormone induces receptor formation or amplification, in other words a hormonal imprinting. Substances acting on the intracellular Ca2+ level of the Tetrahymena, such as TMB-8, EDTA, EGTA, NiCl2 and La(NO3)3, interfered with hormonal imprinting of the unicellular to different degrees, and some of them influenced hormone (insulin, TSH) binding also independently of imprinting. Interference with the intracellular Ca-metabolism generally influenced imprinting by insulin and TSH, which were mediated by different mechanisms, to dissimilar degrees, or in opposite directions. On combined application of the agents acting on Ca-metabolism, their effects were additive. It appears that intact Ca-mediation is an essential prerequisite for normal hormonal imprinting.     

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.     

Imprinting with oxytocin and vasopressin in Chang liver cell cultures: hormonal overlap, binding and influence on cell division

In Chang liver cells the administration of oxytocin and vasopressin as well as the combined application of the two hormones will result in a positive binding imprinting for oxytocin and a negative binding imprinting for vasopressin. The hormones are able to increase the mitotic capacity of the liver cells even without previous imprinting, both in the case of treatment for 4 hours and for 24 hours; the change, however, is more marked in the case of treatment for 4 hours. Treatment for 24 hours results also in some functional imprinting.     

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.     

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.     

Cytoplasmic manifestation of the nuclear membrane's hormone binding capacity during cell division

Summary Binding of insulin and thyrotropic hormone (TSH) to the nuclear membrane of Chang liver cells was demonstrated by qualitative and quantitative cytofluorimetry, which failed to substantiate a similar binding affinity for BSA. It appears that in the dividing cell the binding structures (receptors) of the nuclear membrane migrate in the cytoplasm together with the chromosomes by the end of the prophase and become reorganized in the nucleus around the telophase. The fluorescence which indicated binding also appeared in the midbody region during division of the two daughter cells. These experimental observations strongly suggest that, after cell division, only part of the nuclear membrane's receptor complement has to be resynthesized in the daughter cells, because the receptor number required by a single cell is conserved in cytoplasmic membrane details of nuclear membrane origin.     

Cytoplasmic manifestation of the nuclear membrane's hormone binding capacity during cell division

Binding of insulin and thyrotropic hormone (TSH) to the nuclear membrane of Chang liver cells was demonstrated by qualitative and quantitative cytofluorimetry, which failed to substantiate a similar binding affinity for BSA. It appears that in the dividing cell the binding structures (receptors) of the nuclear membrane migrate in the cytoplasm together with the chromosomes by the end of the prophase and become reorganized in the nucleus around the telophase. The fluorescence which indicated binding also appeared in the midbody region during division of the two daughter cells. These experimental observations strongly suggest that, after cell division, only part of the nuclear membrane's receptor complement has to be resynthesized in the daughter cells, because the receptor number required by a single cell is conserved in cytoplasmic membrane details of nuclear membrane origin.     

Expression and characterization of a functional human insulin-like growth factor I receptor

Stable transfectants of Chinese hamster ovary (CHO) cells were developed that expressed the protein encoded by a human insulin-like growth factor I (IGF-I) receptor cDNA. The transfected cells expressed approximately 25,000 high affinity receptors for IGF-I (apparent Kd of 1.5 X 10(-9) M), whereas the parental CHO cells expressed only 5,000 receptors per cell (apparent Kd of 1.3 X 10(-9) M). A monoclonal antibody specific for the human IGF-I receptor inhibited IGF-I binding to the expressed receptor and immunoprecipitated polypeptides of apparent Mr values approximately 135,000 and 95,000 from metabolically labeled lysates of the transfected cells but not control cells. The expressed receptor was also capable of binding IGF-II with high affinity (Kd approximately 3 nM) and weakly recognized insulin (with about 1% the potency of IGF-I). The human IGF-I receptor expressed in these cells was capable of IGF-I-stimulated autophosphorylation and phosphorylation of endogenous substrates in the intact cell. This receptor also mediated IGF-I-stimulated glucose uptake, glycogen synthesis, and DNA synthesis. The extent of these responses was comparable to the stimulation by insulin of the same biological responses in CHO cells expressing the human insulin receptor. These results indicate that the isolated cDNA encodes a functional IGF-I receptor and that there are no inherent differences in the abilities of the insulin and IGF-I receptors to mediate rapid and long term biological responses when expressed in the same cell type. The high affinity of this receptor for IGF-II also suggests that it may be important in mediating biological responses to IGF-II as well as IGF-I.     

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.     

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.