Tyrosine transaminase induction by dexamethasone in a new rat liver cell line.

A cell line derived from normal, adult rat liver has been established; the cells are similar to hepatocytes, as shown by electron microscopy. The addition of dexamethasone to the culture medium induced a three- to sixfold increase in the specific activity of tyrosine alpha-ketoglutarate transaminase; this increase was inhibited by the simultaneous addition of cycloheximide or actinomycin D. The latter, when added to cells given prior treatment with dexamethasone, further enhanced the transaminase activity. Contact-inhibited cells showed a lower response to dexamethasone than exponentially growing cells.

Early stages of p53-induced apoptosis are reversible.

Apoptosis is a type of physiological cell death that occurs during development, normal tissue homeostasis, or as a result of different cellular insults. The phenotype of an apoptotic cell is relatively consistent in most cases of apoptosis and involves at least changes in the cell membrane, proteolysis of cytoplasmic and nuclear proteins, and eventual destruction of nuclear DNA. Our laboratory is interested in the reversibility of apoptosis. We have initial evidence that DNA repair is activated early in p53-induced apoptosis and may be involved in its reversibility. The present work further strengthens our proposition that p53-induced apoptosis is reversible. We show that p53 activation induces phosphatidylserine (PS) externalization early in apoptosis, and that these early apoptotic cells with externalized PS can be rescued and proliferate if the apoptotic stimulus is removed. In addition, we show that unscheduled DNA synthesis occurs in early apoptotic cells, and that if DNA repair is inhibited by aphidicolin, apoptosis is accelerated. These results confirm that early p53-induced apoptotic cells can be rescued from the apoptotic program, and that DNA repair can modulate that cell death process.

DNA repair is activated in early stages of p53-induced apoptosis.

p53 is a complex molecule involved in apoptosis, cell cycle arrest, and DNA repair. Since apoptosis may play an important role in deletion of neoplastic cells, an understanding of the mechanism of p53-induced apoptosis may be critical for possible future therapeutic interventions. Recent evidence suggests that p53-induced apoptosis may involve members of the nucleotide excision repair (NER) family, linking these two cellular events. Our work using a temperature-sensitive p53 construct further analyzes p53-induced apoptosis in cultured murine mammary epithelial cells and also suggests that DNA repair plays a role in that process. Although p21 is induced in our system, apoptosis occurs without a detectable preceding G1 cell cycle arrest and independent of cellular alterations brought on by the temperature shift. In addition, clonogenic assays suggest that early stages of p53-induced apoptosis may be reversible upon removal of the apoptosis stimulus. As a possible explanation for this reversibility, our results show that general DNA repair activity increases early in p53-induced apoptosis. We also show that caspase-3 is activated at a timepoint when colony formation begins to drop, suggesting a possible mechanism for the point of no return in p53-induced apoptosis.

Regulation of the cell cycle by diethylstilbestrol and progesterone in cultured endometrial cells.

Cultures of primary explanted rabbit endometrium epithelial cells were found to be a mixture of dividing or cycling (20%) and non-dividing or GO (80%) cells. The addition of diethylstilbestrol had the effect of switching most of the GO cells into the cell cycle and to shorten the length of the cell cycle through a shortening of G1 and S. Progesterone had the opposite effects.

Uteroglobin production by cultured rabbit uterine epithelial cells.

Uteroglobin (UG) is a secretory protein produced by the rabbit endometrium and its production is increased during cell differentiation which occurs during early pregnancy or pseudopregnancy. In the present study, the optimal conditions for UG production by rabbit endometrial epithelial cells in culture were examined. Metabolic labeling studies showed the incorporation of [35S]methionine into UG molecules by the endometrial epithelial cells in culture. Accumulation of UG in culture media was linear for at least a period of 24 h. These cells do not catabolize exogenously added radiolabeled UG. Endometrial cells obtained from virgin female rabbits at different times after the administration of human CG (hCG) and put in culture were found to make different amounts of UG. The maximal UG production was found in cells taken from pseudopregnant rabbits 4 days after hCG administration. Cycloheximide (28 micrograms/ml) inhibited the production of UG by the cells in culture whereas actinomycin-D (5 micrograms/ml) and cordycipin (50 micrograms/ml) increased its production. Inhibition of DNA synthesis by hydroxyurea (10(-3) M) did not affect the UG production. The production of UG was significantly less when cells were cultured on attached or floating collagen gels as compared to cells grown on plastic Petri dishes. The amino acid content of Ham's F-12 medium was shown to be adequate for maximal UG production; lowering this amino acid concentration decreased the amount of UG accumulated in the medium over a 24-h period. Increasint the number of cultured cells per dish resulted in an increased UG production per cell.

Estrogen and progesterone regulation of proliferation, migration, and loss in different target cells of rabbit uterine epithelium.

We have explored the possibility that estrogens and progesterone could have different target uterine cell populations according to their cell cycle stage and localization in glands vs. lumen. Experiments were carried out in which rabbits were injected with [3H]thymidine for 3 days to label nuclei of dividing cells, then either 17 beta-estradiol, progesterone, or vehicle were administered. 17 beta-Estradiol induced a decrease in the percentage of cells with labeled nuclei or labeling index of either luminal or glandular epithelium. Since this steroid has been shown to have a significant proliferative effect on glands, the data suggest that its effect is exerted on unlabeled quiescent cells, which are then recruited into the cell cycle. Progesterone, on the other hand, was found to induce a significant increase in labeling index of both luminal and glandular epithelium. Therefore, it is concluded that dividing cells are a target for this hormone. Analysis of the number of nuclear grains according to cell location in luminal vs. glandular epithelia and the effect of hormone administration confirmed that each ovarian hormone acts on different target cell populations. Short and long term administration of estrogens resulted in a larger internal circumference of the uterus due to an increase in the number of luminal cells, whereas the number of glands and glandular cells per section did not appear to change. These findings, in combination with previous research, suggest that endometrial gland cells migrate towards the lumen and estrogen administration decreases the rate of cell loss in the luminal epithelium. The concept of cell migration is supported by experiments in which single administration of [3H]thymidine to rabbits was followed by determination at different times of the geographical distribution of cells with labeled nuclei. There was observed, as a function of time, a decrease in the number of labeled cells in the bottom of the glands with a concomitant increase in the same parameter in the upper part of the glands and luminal epithelia. Estradiol administration changed these kinetics.

Prostaglandin photoaffinity probes: synthesis and biological activity of azide-substituted 16-phenoxy- and 17-phenyl-PGF2 alpha prostaglandins.

The development of a prostaglandin PGF2 alpha photoaffinity probe led to the synthesis and biological evaluation of azide-substituted 17-phenyl-18,19,20-trinorprostaglandin F2 alpha and 16-phenoxy-17,18,19,20-tetranorprostaglandin F2 alpha derivatives. Two approaches for the preparation of iodinated versions of these prostaglandins were evaluated: (1) iodination of a phenyl azide bearing an activating hydroxyl group and (2) iodination of an aniline precursor to the phenyl azide group and subsequent conversion of the aniline to the phenyl azide. In the first approach, 17-(4-azido-2-hydroxyphenyl)-18,19,20-trinorprostaglandin F2 alpha, 16-(5-azido-3-hydroxyphenoxy)-17,18,19,20-tetranorprostaglandin F2 alpha, and 16-(4-azido-2-hydroxyphenoxy)-17,18,19,20-tetranorprostaglandin F2 alpha were prepared by using the Corey synthesis, but were biologically inactive presumably as a result of the hydrophilic phenolic hydroxyl group. In the second approach, the iodination of a 17-(4-aminophenyl)-18,19,20-trinorprostaglandin F2 alpha derivative delivered 17-(4-azido-3-iodophenyl)-18,19,20-trinorprostaglandin F2 alpha, which exhibited competitive binding with natural [3H]PGF2 alpha to ovine luteal cells and to plasma membranes of bovine corpora lutea. [125I]-17-(4-Azido-3-iodophenyl)-18,19,20-trinorprostaglandin F2 alpha was utilized in a preliminary photoaffinity cross-linking experiment.

Effects of 17 beta-estradiol and progesterone on the metabolism of benz[a]pyrene by rabbit endometrial cells in primary culture.

The direct effects of estrogen (17 beta-estradiol) and progesterone on the metabolism of [3H]benzo[a]pyrene (BP) by primary cultures of rabbit endometrium epithelial cells were studied. 17 beta-Estradiol (10(-7) M) did not affect the metabolism of [3H]BP, expressed as the formation of water soluble metabolites of [3H]BP, during a 24-h incubation period. However, progesterone (10(-7) M) inhibited the formation of water-soluble metabolites of [3H]BP and the inhibitory effect was not prevented by 17 beta-estradiol. Progesterone did not appear to affect the relative distribution of the major conjugate types (sulfate esters and glutathiones) that comprised the water-soluble metabolites of [3H]BP. The results, which showed that progesterone acted to inhibit the metabolism of BP by endometrial cells, suggest additional evidence of a role for the ovarian hormone in the modulation of the carcinogenic efficacy of chemical carcinogens in a hormone-responsive tissue.

The biology of apoptosis.

Apoptosis is a complex process that removes aging or injured cells from the body and occurs in a wide variety of organisms. Cell death has always been an integral aspect of the study of pathology, but only over the last 30 years or so has the interest in apoptosis gained appreciation in this field. This review analyzes pertinent aspects of apoptosis, from Virchow's initial descriptions of necrobiosis to more modern research, and reviews some of the key events and molecules involved in the process. Finally, the role of apoptosis in certain diseases and its importance in the clinical setting is addressed.

Triacetylated insulin: biologic activity and resistance to degradation.

Tritiated N-hydroxysuccinimide acetate was prepared with specific activities up to 5 Ci/mmole and utilized to prepare tritiated triacetyl insulin. Binding of triacetyl insulin to liver plasma membranes was measured by its capacity to displace 125I-monoiodoinsulin. At low concentrations, less than 10 ng/ml triacetyl insulin appears to be as effective as native insulin in reducing the binding of 125I-monoiodoinsulin to plasma membranes. At concentrations of 20 ng/ml and higher, triacetyl insulin is significantly less effective than native insulin in displacing binding of 125I-monoiodoinsulin to plasma membranes. The properties of triacetyl insulin in this system are not ascribable to deacetylation and conversion of the substituted product to native insulin. Biologic activity of triacetylated insulin was studied in two other in vitro systmes. A comparison was made of the capacity of native beef insulin and its triacetyl derivative to stimulate glucose oxidation by epididymal fat pads. At all three concentrations tested (2, 6, and 18 ng/ml), triacetyl insulin exerted considerable activity, although its potency was significantly less than that of native insulin. Similar effects were observed when biologic activity was measured by induction of tyrosine-alpha-ketoglutarate transaminase in a cultured liver cell system where significant activity of triacetyl insulin was found at concentrations of 10(-9)-10(-7) M. In all systems tested, the activity of triacetylated insulin could not be accounted for by deacetylation and conversion to native insulin. In all systems studied, triacetyl insulin was more resistant to degradation than was monoiodoinsulin.

Effect of phorbol ester on prostaglandin regulation of proliferation in rabbit endometrial cells.

We have proposed that two of the endogenously synthesized endometrial prostaglandins, prostaglandin F2 alpha (PGF2 alpha) and prostaglandin E1 (PGE1), play a regulatory role in growth control of the endometrium. PGF2 alpha increases DNA synthesis and PGE1 inhibits that effect. Primary cultures of rabbit endometrial cells were used here to examine the effects of the tumor-promoting, diacylglycerol mimicking, phorbol ester, 12-O-tetradecanoyl phorbol-13-acetate (TPA), on the prostaglandin control of cell proliferation. TPA treatment of these cultures results in: a decrease in control levels of proliferation and complete inhibition by TPA of PGF2 alpha stimulated DNA synthesis; a reduction in [3H]PGF2 alpha binding with short term treatment but an increase to above control binding level with long term treatment; an inhibition of the normal PGF2 alpha stimulated inositol polyphosphate synthesis; and a small increase in accumulation of PGF2 alpha in the culture media. Furthermore, in this culture system, TPA does not down regulate [3H]PGE1 binding; it does not alter the normal PGE1 stimulation of cAMP synthesis; and it has no effect on the normal endogenous PGE1 synthesis by these cultures. The above results are consistent with our previous observations that PGF2 alpha works through the intracellular messengers inositol polyphosphate/diacylglycerol whereas PGE1 works through cAMP.

A developmental view of endometrial hyperplasia and carcinoma based on experimental research.

On the basis of experimental research, using rabbit uterine epithelium as a model, it is postulated that human endometrial hyperplasia or carcinoma may be due to derangement of intrinsic growth mechanisms such as cell proliferation, migration, loss and differentiation. Ovarian hormones, estrogen inhibitor or amplifying factors, prostaglandins, stroma-epithelial interactions, proteolytic activity and hormone receptors, all regulate the described intrinsic growth mechanisms, and their excess or lack could result in altered growth patterns. It is also proposed that different types of endometrial carcinoma could result from neoplastic transformation of cells at different stages of differentiation. Since cells at those stages could respond to various hormones in different ways, it would seem of therapeutic value to know the cell of origin in each type of endometrial carcinoma.

A role for prostaglandins in estrogen growth regulation.

Several lines of evidence support the theory that estrogen increases prostaglandin F2 alpha (PGF2 alpha) receptor number and amplifies the intracellular response to the uterine mitogen, PGF2 alpha, in primary cultures of rabbit endometrium. It is proposed here that estrogen induction of growth in target tissues may be mediated through prostaglandins. Determination of prostaglandin receptor status and prostaglandin levels in estrogen dependent tumors could be of importance as diagnostic tools. The use of prostaglandin synthesis inhibitors and/or antagonists in the therapy of these tumors should be considered.