Coordinated modulation of albumin synthesis and mRNA levels in cultured hepatoma cells by hydrocortisone and cyclic AMP analogs.

The treatment of Hepa-2 cells, a permanent mouse hepatoma cell line, for 72 h with hydrocortisone (10(-6) M), N6,O2-dibutyryl cyclic AMP (10(-3) M), or 8-bromocyclic AMP (10(-3) M) results in a 2-,3- or 4-fold increase, respectively, in rates of synthesis and secretion of mouse serum albumin. Simultaneous treatment with hydrocortisone and N6,O2-dibutyryl cyclic AMP results in a 10-fold stimulation in these parameters, an effect that is significantly more than additive for the two compounds tested. The number of albumin mRNA sequences, determined by hybridization of total cell RNA to albumin complementary DNA, was increased in direct proportion to the increases in albumin synthesis in all experiments. The relative rate of albumin synthesis approaches in vivo levels in cells treated simultaneously with hydrocortisone and N6,O2-dibutyryl cyclic AMP. We propose that these factors may be necessary to maintain the maximal level of differentiated function in the continuous culture of Hepa-2 cells.     

Effect of hydrocortisone on superoxide radical production by phagocytosing spleen cells

Superoxide radical production by mouse phagocytic spleen cells was shown to be essentially increased 2 hours following intraperitoneal injection of hydrocortisone acetate at a dose of 50 mg/kg body weight. The addition of hydrocortisone to the suspension of mouse spleen cells has resulted in linear dependence of hormone concentration in the incubation medium on the maximum rate of superoxide radical production. The mechanism of hydrocortisone stimulating effect on the activity of plasma membrane-located NAD(P)H-oxidase of phagocytic cells is being discussed.     

Interaction of chalones and glucocorticoid hormones in regulation of cell division

The action of hepatic chalone on cell proliferation in inoculated hepatoma 22a of mice was studied in the presence of a changed level of glucocorticoid hormones in experimental animals. Chalone was obtained from the liver of intact rats by ethanol precipitation. The intensity of cell proliferation in hepatoma was evaluated by the colcemide and autoradiography methods. Six hours after chalone injection c-mitosis in the tumor decreased 2.7-fold, and the DNA index 6.8-fold. It may be concluded that the preparation used contains both G1- and G2-chalones. Single or repeated injections of hydrocortisone to mice inhibits cell proliferation to a less degree than administration of chalone alone. Combination of hydrocortisone and chalone produces the same effect as injection of chalone alone. Adrenalectomy diminishes susceptibility of hepatoma cells to exogenous chalone. The degree of tumor proliferative activity in the adrenalectomized animals was half as much after chalone injection, as compared to that in intact animals. Thus, a certain level of glucocorticoid hormones in hepatoma tissue is necessary to reveal the action of chalones.     

Changes in the H-alloantigen-recognizing function of mouse lymphoid cells following hydrocortisone administration]

The capacity of spleen, thymus, and bone marrow cells of intact (control) and of hydrocortisone-treated mice CBA to induce the lymph node type of graft-v-host reaction (GVHR) in hybrids F1 (CBA X c57bl) was studied. After hydrocortisone injection (2.5 mg per mouse) the donor spleen cells became more active in GVHR, considering the value of lymph node indices and immunoblast content in the regional lymph node as compared with a control group. Following transplantation of thymus cells taken from the hydrocortison-treated donors the immunoblast count was higher, although the lymph node weight remained the same as in the control group. On the contrary, following the transfer of the bone marrow cells from the hydrocortisone-treated mice the lymph nodes enlarged, while the immunoblast count remained as low as in control. Consequently, exogenously conditioned increase in the hydrocortisone level was accompanied by an enrichment of the spleen and thymus cell populations with T-lymphocytes, proliferating in response to H-alloantigens.     

Glucocorticoids enhance viral transformation of mammalian cells

The effect of glucocorticoids on the replication of murine sarcoma virus (MSV) in mammalian cells were examined. Glucocorticoids, hydrocortisone (1 to 10 micrograms/ml) and dexamethasone (5 micrograms/ml), enhance transformation induced by the Kirsten strain of MSV (Ki-MSV) in normal rat kidney and human cells 10- to 30-fold. The enhancing effect was much more pronounced in normal human colonic mucosal epithelial-like cells. On the other hand, the hormones estradiol, testosterone, and progesterone had no effect (5 micrograms/ml). Individual foci appeared earlier and were larger in hydrocortisone-treated cells compared with untreated cells. This enhancing effect is further evidenced by the increased virus yield and murine leukemia virus complement-fixing antigen production in the test system. However, such enhancement of hydrocortisone on the Ki-MSV-induced transformation was not observed in mouse embryo cells as previously reported.     

Localization of the GLUT1 glucose transporter to brefeldin A-sensitive vesicles of differentiated CIT3 mouse mammary epithelial cells

Glucose is a precursor of lactose, the major carbohydrate and osmotic constituent of human milk, which is synthesized in the Golgi. The GLUT1 glucose transporter is the only glucose transporter isoform expressed in the mammary gland. The hypothesis that lactogenic hormones induce GLUT1 and cause its localization to the Golgi of mammary epithelial cells was tested in CIT(3)mouse mammary epithelial cells. Treatment with prolactin and hydrocortisone caused a 15-fold induction of GLUT1 by Western blotting, but 2-deoxyglucose uptake decreased. Subcellular fractionation and density gradient centrifugation demonstrated enrichment of Golgi fractions with GLUT1. Lactogenic hormones enhanced GLUT1 glycosylation, but did not determine whether GLUT1 was targeted to plasma membrane or to Golgi. Confocal microscopy revealed that lactogenic hormones alter GLUT1 targeting from a plasma membrane pattern to a predominant perinuclear distribution with punctate scattering through the cytoplasm. GLUT1 is targeted to a compartment which is more sensitive to Brefeldin A than the compartments in which GM130 and beta-COP reside. Targeting of GLUT1 to endosomes was specifically excluded. We conclude that prolactin and hydrocortisone induce GLUT1, enhance GLUT1 glycosylation, and cause glycosylation-independent targeting of GLUT1 to Brefeldin A-sensitive vesicles which may represent a subcompartment of cis-Golgi. These results demonstrate a hormonally-regulated targeting mechanism for GLUT1 and are consistent with an important role for GLUT1 in the provision of substrate for lactose synthesis.     

In vitro studies on lymphocyte differentiation. II. Generation of terminal deoxynucleotidyl transferase-positive cells in long-term cultures of mouse bone marrow.

The enzyme TdT is the earliest known marker of lymphocytic differentiation in rodents. Cells containing this enzyme were demonstrated in suspension cultures of mouse bone marrow cells that had been maintained in vitro for periods of 7 to 45 days. The cells were detected by immunofluorescence using purified antibodies to homogeneous TdT. Between 0.04 and 2.0% of cultured bone marrow cells from a variety of mouse strains were positive. More than 40% of the TdT-positive cells incorporated 3H-thymidine during a 20-min pulse. Surface Ig and Thy-1 antigens were not detected on the TdT-positive cells. The prevalence of TdT-positive cells was decreased 10-fold in cultures that had been treated with 10(-6) M hydrocortisone 24 hr before harvesting. The results indicate that lymphoid progenitor cells can be generated in vitro.     

Study of rat pulmonary macrophages as affected by hydrocortisone administration and adrenalectomy

The blood clearance rate of inert colloidal particles and the number of rat lung interstitial phagocytic cells decrease considerably on the 7th day after daily subcutaneous hydrocortisone acetate (HC) injection at a dose of 125 mg/kg. The number of cells in the bronchoalveolar lavage (BAL) increases more than 5-fold, and the absolute quantity of neutrophils is 66 times higher than in the control. Phagocytic and microbicidal activity of HC-treated animal alveolar macrophages (AM) decreases. Stimulation with zymosan led only to the recovery of the normal parameters of mononuclear phagocytosis system (MPC) and its pulmonary compartment activity. The parameters of MPS and AM studied increase on the 7th day after bilateral adrenalectomy (AE). The number of BAL cell increases 1.4-fold due to the animals' death immediately after intravenous zymosan injection because of total hemorrhage. The data obtained testify to the influence of glucocorticoids on the composition and activity of bronchoalveolar space cells, which in turn determine the resistance of the lung tissue.     

Influence of Osteoprotegerin (OPG) on Resorption of Heterotopically Induced Ossicle

In this paper, the effect of osteoprotegerin (OPG) on slowing down the resorption process of heterotopically induced bone tissue is described. The induced ossicle is resorbed ex inactivitate. This system mimics osteoporosis in immobilised skeletal bones. Bone induction was achieved in BALB/c mice after the injection of the suspension of 3 x 10(6) HeLa cells into thigh muscle of animals immuno-suppressed by a single dose of hydrocortisone. To slow down the process of resorption we applied OPG and measured quantitatively the effect by weighing the mass of mineral deposited in the induced ossicle after hydrolysis of soft tissues surrounding the induced ossicles. As the effect of application of OPG more than 340-540% of bone mineral is found in the induced ossicles following nine applications of 0.05 mg OPG per mouse, every second day--in comparison to the control animals.     

Changes in the mitotic activity and destruction of rat thymus lymphocytes following hydrocortisone administration at different hours of the day]

The 24-hour rhythm of mitoses was identical in the thymus lymphocytes of 30-day rats in control and in experimental animals 4 hours after injection of hydrocortisone. In the control rats the number of degenerating lymphocytes failed to alter in the course of 24 hours. Four hours after the hydrocortisone injection of degenerated cells increased sharply; however, the rate of the lymphocyte destruction was more significant at night and early in the morning than during the day and the evening.     

Effect of adrenalectomy of the recipients of allogeneic lymphocytes on the inactivation of endogenous colony-forming cells in mice

Intravenous injection of lymph node cells from the parental C57BL/6 mouse line in doses of 2 X 10(6) or 5 X 10(6) into sublethally irradiated (CBA X C57BL/6) F1 hybrids produced more demonstrable suppression of endogenous colony-formation in adrenalectomized recipients as compared with that seen in sham-operated on ones (P less than 0.01). The recipients' adrenalectomy itself was accompanied by an over 2-fold increase in the number of the endogenous colony-forming cells in the spleen as compared with sham-operated on mice. Possible mechanisms, by which the killer action of lymphocytes on the endogenous colony-forming cells is potentiated, are under discussion.     

Precocious decrease of chymotrypsinogen by thyroxine in the pancreas of suckling mice

Chymotrypsinogen activity in mouse pancreas gradually decreased from birth, reaching the adult level on day 20 after birth. In suckling mice the enzyme activity was decreased to 1/9 of the control value by injection of thyroxine. The activity was not affected by insulin, and was slightly increased, rather than decreased, by daily injection of hydrocortisone. The effect of thyroxine seemed to be direct, not due to modification of adrenal function.     

Precocious induction of pepsinogen in the stomach of suckling mice by hormones

Effects of hormones on pepsinogen activity in mouse stomach were investigated by enzyme assay and electron microscopy. Administration of hydrocortisone alone to mice on days 5–10 increased the enzyme activity in the stomach to as much as 4.5-fold that of untreated mice and the increase was dose dependent. Thyroxine also evoked precocious differentiation of the stomach. The effects of thyroxine and hydrocortisone were additive. Injections of insulin had little effect when given alone, or in combination with other hormones. Injection of hydrocortisone alone or plus thyroxine also caused morphological differentiation of the chief cells in the stomach mucosa. Administration of thyroxine to mice on days 15–20 induced as much enzyme activity as that induced by hydrocortisone, but neither of these hormones had any effect when injected after day 23.These results suggest that besides hydrocortisone, thyroxine is also involved in differentiation of the stomach in mice for the first 20 days after birth and that the normal increase of pepsinogen activity in the stomach of mice during the late suckling period is brought about by serum glucocorticoids, possibly with thyroxine.     

Characteristics of the reactions to N-alloantigens of lymphoid cells from hydrocortisone-stimulated and adrenalectomized mice]

The capacity of the spleen, bone marrow and thymus cells from CBA mice (intact, adrenalectomized, and those treated with single or repeated hydrocortisone injections) to induce the lymph node type of "graft-versus-host" reaction (GVHR) in (CBA X C57BL) F1 hybrid recipients was evaluated. Two days after 2.5 mg hydrocortisone injection the capacity of the spleen and bone marrow cells to induce GVHR increased while that of the thymus cells remained unchanged. Seven and particularly 15 days after hydrocortisone injection the spleen cells became less active. Two days following repeated daily hormone injections in a dose of 0.25 mg within 18 days the thymocyte activity in GVHR increased, while that of the spleen and bone marrow cells did not change.     

A study of the frequency of sister-chromatid exchange and of thioguanine-resistant cells in mouse spleen lymphocytes after in vivo exposure to ethylnitrosourea

As part of the development of a multi-endpoint, in vivo, mouse model for mutagenesis we have measured the frequency of sister-chromatid exchange (SCE) and the frequency of thioguanine-resistant (TGr) cells among the lymphocytes of the mouse spleen following acute, intraperitoneal exposure to ethylnitrosourea (ENU). The responses of these two endpoints have been monitored both as a function of the dose of ENU injected, ranging from 0 to 70 mg/kg, and as a function of time after injection, from 1 day to 72 days. The SCE frequency response was highest 1 day after the ENU was injected, increasing 2.5-fold over control values for mice that received 70 mg/kg, and declined to control values in all animals by 72 days. SCE showed a linear dose response both at 1 day and 8 days after injection. The frequency of TGr cells was at control levels at 1 day, but at 15, 36 and 72 days after ENU injection the frequency of TGr cells showed a linear dose response. In addition, the frequency of TGr cells increased linearly with time for both the 35 and 70 mg/kg doses. The frequency of TGr cells for mice that had received 70 mg ENU/kg 72 days previously, was 100-fold higher than in control animals, giving a frequency of 1.4 X 10(-4).     

Regulation of collagenase production by steroids in uterine smooth muscle cells: An enzymatic and immunologic study

An enzyme-linked immunosorbent assay (ELISA) has been developed for rat collagenase. The assay is capable of measuring the enzyme from a variety of rat cell sources at concentrations of 10–;50 ng/ml, approximately 500–;1,000-fold more sensitive than radiolabelled collagen fibril assay systems. The assay is specific to collagenase from the rat: enzymes from human, tadpole, mouse, and bacterial sources failed to cross-react significantly with rat enzyme. The assay is reproducible and accurate, and is capable of detecting enzyme in the presence of serum or tissue inhibitors. Using the ELISA, we have examined the effect of a variety of hormones on the production of collagenase by rat myometrial smooth muscle cells in culture. Of all the reproductive hormones examined, only progesterone and its synthetic derivative medroxyprogesterone acetate were capable of inhibiting the production of the enzyme by these cells. The maximally effective concentration of progesterone was 1 x 10^?6M, and that of medroxyprogesterone acetate was 1 x 10^?7M. The effect of the steroid was selective: no effect on cell proliferation or on general protein synthesis was observed. In addition to the progestational steroids, the glucocorticoids were also capable of inhibiting the production of collagenase by the cells at similar nominal concentrations. However, the myometrial cells were found actively to metabolize progesterone but not hydrocortisone in culture. Thus, the effective inhibitory concentration of progesterone was approximately ten-fold lower than that of hydrocortisone. The results of this study support the concept that progesterone plays a major role in preventing the production of collagenase in the rat uterus.     

Serum lysozyme in mice subjected to combined immunosuppression with 6-mercaptopurine and hydrocortisone

Simultaneous peroral administration of 6-mercaptopurine (80 mg/kg per day) and subcutaneous injection of hydrocortisone (1 mg/mouse per day) for ten days results in increased lethality and more pronounced decrease in total peripheral leukocyte count and serum lysozyme levels as compared with mice receiving each drug separately. The possible mechanism of this effect is discussed.     

Change in the absorptive capacity of macrophages from different organs after administration of hydrocortisone

Two, twenty-four and 48 h after hydrocortisone treatment in a dose of 125 mg/kg bw the blood clearance rate for colloidal carbon particles in rats turned to be 2, 2.1. and 1.6 times less whereas that for 51Cr-SRBC in CBA mice 2.1, 2.2 and 1.7 times less as compared to untreated controls. Within 24 and 72 h after hormone injection the efficacy of red blood cell uptake by Kupffer cells decreased 1.35 and 1.8 times whereas the similar uptake by lung or spleen macrophages changed but insignificantly and that by bone marrow cells was even greater than in controls. Toward the 5th day after zymosan treatment the uptake capacity of Kupffer cells was the greatest whereas the plasma 11-OHCS content was 1.3-fold less versus the control values.     

Interleukin-1 receptor antagonist binds to the type II interleukin-1 receptor on B cells and neutrophils.

We have examined the binding of human and rodent interleukin-1 receptor antagonist (IL-1ra) to the type II IL-1 receptor on the human B cell line, Raji, on the mouse pre-B cell line, 70Z/3, and on human polymorphonuclear leukocytes (PMNs). Human IL-1ra binds to the receptors on the human B cells with an affinity (KD = 15 +/- 3 nM) equal to that of IL-1 alpha and only 15-fold lower than that of IL-1 beta and, likewise, binds to human PMNs with an affinity (KD = 8 +/- 4 nM) 15-fold lower than that of IL-1 beta. Mouse and rat IL-1ra bind to these two human cell types with an affinity similar to that of the human protein. Human IL-1ra binds very weakly to the type II receptor on the mouse pre-B cells with an affinity (KD = 1.4 +/- 0.2 microM) about 1500-fold lower than human IL-1 beta. Mouse and rat IL-1ra also bind to the mouse pre-B cells with low affinity. The weak binding of the three IL-1ra proteins to these mouse cells appears to be more a consequence of the cell type rather than species specificity. There may be a population of cells for which the actions of IL-1 cannot be effectively opposed by IL-1ra, although this group does not include mature B cells and PMNs.