Microtubule and plasmalemmal reorganization: acute response to estrogen.

The acute ultrastructural effects of estrogen in endometrial epithelial cells were investigated by transmission electron microscopy (TEM), with special reference to the microtubule (MT) apparatus and the luminal surface. Ovariectomized rats anesthetized with pentobarbitol sodium were injected intravenously with estradiol-17 beta (E2 beta), 0.5 micrograms/100 g body wt. At intervals from approximately 30 s to 30 min thereafter, 70-80 nm cross sections of a uterine horn were prepared for TEM. In placebo controls, cytoplasmic MT were conspicuous in length and number, whereas only a minimal population of short microvilli (MV) was evident. In contrast, the specimens subjected to E2 beta for only 35 s showed a significant decrease in MT number and length, with virtually complete depletion of these organelles by approximately 80 s. Concomitantly, the luminal MV exhibited striking enhancement in length and density. Thereafter, these rapid and reciprocal alterations of MT and MV underwent inversion. Thus MT structures began to reappear within 2 min, increasing progressively so that by 30 min their numbers were again substantial, although lengths remained diminished. During the same interval, the initial surge of luminal MV gradually subsided, to near-control appearance by 30 min. These coordinate, reciprocal, and biphasic responses are consistent with biochemical evidences of abrupt membrane perturbation associated with interception of estrogen at its cellular targets. The resultant modification of the intracellular environment may contribute to limited reorganization of cellular architecture and propagation of the hormonal signal.

Characterization and partial purification of the Drosophila Kc cell ecdysteroid receptor.

The molting hormones of insects, the ecdysteroids, are steroids whose action is mediated by an intracellular receptor. The Kc cell line of Drosophila melanogaster possesses ecdysteroid receptors and exhibits characteristic, receptor-dependent morphological and biochemical responses to the application of ecdysteroids. This paper describes the interaction of muristerone A (2 beta, 3 beta, 5 beta, 11 alpha, 14 alpha(20R,22R)- heptahydroxycholest-7-en-6-one), a phytoecdysteroid, with the Kc cell ecdysteroid receptor. Muristerone A-receptor complexes are not as sensitive to dissociation in high salt buffers as other ecdysteroid-receptor complexes we have examined. This has enabled us to use [3H]muristerone A to follow the Kc cell ecdysteroid receptor during heparin-agarose, DNA-cellulose, and hydroxylapatite chromatography, as well as gel filtration and ion exchange high pressure liquid chromatography. The Drosophila Kc cell ecdysteroid receptor has a Stokes radius of 4.6 nm, a frictional coefficient of 1.4, and a molecular weight of 120,000. A procedure is presented that results in a 750-fold enrichment of the receptor.

Monoclonal antibody toward lysosomal cathepsin B cross-reacts preferentially with distinct histone classes.

1. A set of monoclonal antibodies (Mab) was prepared against cathepsin B (CB) from rat preputial-gland, an organ characterized by rapidly-renewing cell populations, which is a uniquely enriched source of lysosomal enzymes, including CB. Minute amounts of CB are known to be transferred abruptly to the nuclear compartment in a variety of activated cells. 2. Since, on the basis of its stringent substrate requirements, CB was expected to function at limited protein loci in chromatin, Mab Line II-B4 was used to probe Western blots of chromatin fractions and selected proteins. 3. The Mab, which was not directed against the active site of CB, cross-reacted preferentially with histones 3 and 4 (H3 and H4) in acid-soluble fractions of chromatin from rat preputial-gland. Line II-B4 also recognized H3 and H4 selectively in calf thymus histones and among histones purified from a wide range of sources from yeast to man. HMG 1 was minimally immunoreactive among preputial gland constituents and carbonic anhydrase (CA) was also sensitive to the Mab. 4. The common determinants were not shared by any of the H1 series, nor by H2A, H2B, protein A24 or a wide range of natural and synthetic products. 5. Origin of the antigenicity was traced by chemical modifications of H3, H4 and CA to the critical contribution of arginine and hydrophobic amino acid residues in its immediate environment, indicating that Line II-B4 may be directed against an epitope comprising the specific binding-site of CB and its selective substrate(s). 6. These data suggest that certain highly conserved cellular constituents may be uniquely vulnerable to limited proteolysis in preproliferative cells responding to mitotic signals.

Antibodies against estradiol-binding lysosomal lipoproteins gain access to the nuclear compartment of preputial gland cells under estrogen influence.

The influence of estrogen in provoking nuclear recompartmentation of lysosomal components in hormone-sensitive cells was investigated by immunological analyses of isolated nuclei from the preputial glands of ovariectomized rats. Fixed smears were prepared from ultrapurified nuclei freed of outer membrane, 2-30 min after iv injection of placebo-control solution or submicrogram amounts of estradiol-17 beta. Cytoplasmic contamination was negligible in such preparations, as monitored by vital staining with acridine orange. On challenge with immunoglobulin G directed toward a group of lysosomal high density lipoproteins which have been shown to bind estradiol-17 beta specifically, control preparations exhibited minimal indirect immunofluorescence that was essentially confined to the nuclear periphery. In contrast, a high proportion of the nuclei exposed for as little as 2 min to estradiol-17 beta but not to the relatively inert 17 alpha-congener, displayed generally more intense immunofluorescence which was distributed over the entire organellar area. Thus, the immunoglobulin becomes accessible to the nuclear interior in vitro as a function of pretreatment in vivo with active hormone. Nuclei from estrogen-pretreated rats were more structurally labile than corresponding controls, as judged by morphological criteria, even when isolated by gentle teasing rather than subjection to the rigorous ultrapurification process. By either method, integrity of the specimens was enhanced somewhat when they were prepared from rats ovariectomized before experiencing even a single estrous cycle. The observations verify and extend independent biochemical and ultrastructural evidence that structural labilization of cellular organelles and enhanced accessibility of limited amounts of lysosomal constituents to the nuclear compartment of specific target cells are early correlates of estrogen action.

Lysosomal cathepsin B-like activity: mobilization in prereplicative and neoplastic epithelial cells.

Extracellular release of acid thiol proteinase activity by prereplicative and neoplastic epithelial cells was studied in serum-free, chemically defined media (CDM) in vitro. Cells isolated from urinary bladder of male bullfrogs and endometrium of ovariectomized rats each showed preferential secretion of cathepsin B-like (CB) activity within 30 min after exposure to carcinogenic nitrosamines (5 X 10(-4) M) or to mitogenic estrogen 1 X 10(-9) M), respectively. In contrast, release of such proteinase, and stimulation of cell proliferation were far less extensive in rat preputial gland cells treated with estradiol-17 beta. Striking secretion of CB was characteristic of neoplastic, but not noncancerous, epithelial cells from human ectocervix. Neoplastic cells with divergent rates of cell-to-cell aggregation were separated by a filtration method. Those cells with high rates of intercellular aggregation also exhibited higher rates of cell proliferation in CDM, as well as in soft gels, and a greater level of CB release than corresponding cancer cells with a relatively low degree of intercellular adhesion. Brief treatment of neoplastic cervical epithelial cells with liposomes containing entrapped leupeptin, a potent inhibitor of CB activity, elicited a sharp reduction in both cellular thiol proteinase activity and cell growth as compared to appropriate controls. These data indicate that mobilization of lysosomal CB activity in prereplicative and malignant cells may play a significant role in the promotion of cell proliferation.

Elevated serum cathepsin B1 and vaginal pathology after prenatal DES exposure.

Activities of the lysosomal enzymes, cathepsin B1 (CBI), beta-glucuronidase, and beta-N-acetyl-D-glucosaminidase, as well as sialyl transferase, alkaline phosphatase, and placenta-like alkaline phosphatase, were determined on blind-coded serums from 99 women exposed to diethylstilbestrol (DES) in utero and 40 unexposed subjects of comparable age range. Cathepsin B1 averaged 100%, 1040% (P less than 0.001), 2720 % (P less than 0.001), and 4760% (P less than 0.001) of controls in DES-exposed women with no genital tract abnormalities (N = 11), adenosis (N = 68), adenosis with concomitant dysplasia (N = 15), and clear-cell adenocarcinoma (N = 5), respectively. The later two groups also exhibited 0.01). Activities of the other four enzymes in serums of DES-exposed women were unchanged from those controls, suggesting that alterations in CBI were not due to generalized increases in lysosomal membrane instability or other gross cellular damage. In 2 DES-exposed women with clear-cell adenocardinoma, from whom serial samples were available, preoperative levels of serum CBl fell from a mean of 4280% to values indistinguishable from controls by 7--12 days after tumor excision, concurrently with objective signs of remission. Recrudescence of serum CBI levels preceded by at least 3 months clinical evidence of persistent adenosis accompanied by vaginal dysplasia. Although the nature of the increments in CBI-like activity in the majority of subjects with DES-related pathology remains to be determined, the findings may complement present methods of physical diagnosis and prognosis.

The lysosomal membrane complex. Focal point of primary steroid hormone action.

At short intervals after the intravenous administration of oestradiol-17beta, diethylstilboestrol, testosterone or saline control solution to ovariectomized rats, highly purified lysosome samples were prepared in substantial yield from preputial glands, sex accessory organs rich in these organelles. The preparations were essentially devoid of mitochondrial contamination. Exposure in vivo to doses of these hormones varying from 0.1 to 5mug/100g body wt. provoked dose-dependent labilization of the lysosomal membrane surface, as evidenced by significantly diminished structural latency of several characteristic acid hydrolases, including acid phosphatase, beta-glucuronidase and acid ribonuclease II, when such preparations were subsequently challenged in vitro with autolytic conditions, detergent or mechanical stress. Enhanced lytic susceptibility induced by hormone pretreatment was occasionally detectable in the initial preparation without further provocative stimuli in vitro. Comparable results were obtained with the corresponding fractions of uterus, despite the more limited concentration of lysosomes in this steroidal target organ. By the present criteria oestradiol-17alpha was essentially inert, even in a dose 25 times that effective for its active beta-epimer (<0.1mug/100g body wt.). Pretreatment with diethylstilboestrol exerted substantial membrane-destabilizing influence in preputial-gland lysosome samples from orchidectomized rats. Moreover, administration of testosterone to gonadectomized animals resulted in essentially equivalent dose-dependent augmentation of lysosomal enzyme release in preputial-gland preparations of either sex. The membrane stability of lysosome-enriched preparations from uterus, on the other hand, was unaffected by testosterone pretreatment. The sensitivity, specificity and selectivity of the lysosomal response to sex steroids provide evidence for the physiological significance of this phenomenon as a general mechanism for mediation of secondary biochemical transformations in the hormone-stimulated target cell.