Structure and dynamic properties of a glucocorticoid receptor-induced chromatin transition.

Activation of the mouse mammary tumor virus (MMTV) promoter by the glucocorticoid receptor (GR) is associated with a chromatin structural transition in the B nucleosome region of the viral long terminal repeat (LTR). Recent evidence indicates that this transition extends upstream of the B nucleosome, encompassing a region larger than a single nucleosome (G. Fragoso, W. D. Pennie, S. John, and G. L. Hager, Mol. Cell. Biol. 18:3633-3644). We have reconstituted MMTV LTR DNA into a polynucleosome array using Drosophila embryo extracts. We show binding of purified GR to specific GR elements within a large, multinucleosome array and describe a GR-induced nucleoprotein transition that is dependent on ATP and a HeLa nuclear extract. Previously uncharacterized GR binding sites in the upstream C nucleosome region are involved in the extended region of chromatin remodeling. We also show that GR-dependent chromatin remodeling is a multistep process; in the absence of ATP, GR binds to multiple sites on the chromatin array and prevents restriction enzyme access to recognition sites. Upon addition of ATP, GR induces remodeling and a large increase in access to enzymes sites within the transition region. These findings suggest a dynamic model in which GR first binds to chromatin after ligand activation, recruits a remodeling activity, and is then lost from the template. This model is consistent with the recent description of a "hit-and-run" mechanism for GR action in living cells (J. G. McNally, W. G. Müller, D. Walker, and G. L. Hager, Science 287:1262-1264, 2000).

Predictors of complications and learning curve using the Angio-Seal closure device following interventional and diagnostic catheterization.

A retrospective study was conducted to determine the frequency and nature of groin complications when the Angio-Seal device was used on 252 occasions by one operator immediately following interventional (66%) and diagnostic (34%) procedures. Sixty-nine percent of the 238 successfully deployed cases received ticlopidine or clopidogrel, 16% received abciximab, and 15% received heparin postprocedure. Complications included vascular surgery for collagen plug perforation into the femoral artery (0.8%), failure to deploy (5.6%), pseudoaneurysm (0.4%), brisk, visible bleeding (9%), persistent ooze (14%), hematoma > 6 cm (0.8%), hematoma 1 cm(2) (10%). Multivariate analysis identified diagnostic cases (6 Fr sheaths) to be associated with a reduced risk of complications [odds ratio (OR) 0.1] while interventional procedures (8 Fr sheaths), postprocedure heparin, and body mass index (BMI) < 28 (OR 10.1, 3.2, and 2.8, respectively) were associated with increased risk. Gender, age, ticlopidine, clopidogrel, and abciximab were not independent predictors of complications. A learning curve for device deployment was observed in the first 50 cases (14% nondeployment vs. 3.5% for the subsequent 202 procedures, P = 0.009) and failure to deploy was independent of sheath size used. Angio-Seal can be used with reasonable safety and efficacy immediately after diagnostic and interventional procedures. Cathet. Cardiovasc. Intervent. 48:162-166, 1999.

The glucocorticoid receptor is tethered to DNA-bound Oct-1 at the mouse gonadotropin-releasing hormone distal negative glucocorticoid response element.

An element required for glucocorticoid repression of mouse gonadotropin-releasing hormone (GnRH) gene transcription, the distal negative glucocorticoid response element (nGRE), is not bound directly by glucocorticoid receptors (GRs) but is recognized by Oct-1 present in GT1-7 cell nuclear extracts or by Oct-1 purified from HeLa cells. Furthermore, purified full-length GRs interact directly with purified Oct-1 bound to the distal nGRE. Increasing the extent of distal nGRE match to an Oct-1 consensus site not only increases the affinity of Oct-1 binding, but also alters the conformation of DNA-bound Oct-1 and the pattern of protein DNA complexes formed in vitro with GT1-7 cell nuclear extracts. In addition, the interaction of purified GR with DNA-bound Oct-1 is altered when Oct-1 is bound to the consensus Oct-1 site. Mutation of the distal nGRE to a consensus Oct-1 site is also associated with reduced glucocorticoid repression in transfected GT1-7 cells. Furthermore, repression of GnRH gene transcription by 12-O-tetradecanoylphorbol-13-acetate, which utilizes sequences that overlap with the nGRE, is reversed by this distal nGRE mutation leading to activation of GnRH gene transcription. Thus, changes in the assembly of multi-protein complexes at the distal nGRE can influence the regulation of GnRH gene transcription.

Purification and stabilization of transcriptionally active glucocorticoid receptor.

A major obstacle to the purification of glucocorticoid receptor (GR) is the very high nonspecific surface adsorption of this protein. This phenomenon is a property of the GR itself and does not reflect overall protein concentration or buffer conditions. We have observed that the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS) is unique in its ability to stabilize the receptor and largely eliminate loss to nonspecific adsorption. We have coupled this observation with a two-step purification method that allows efficient purification and stabilization of transcriptionally active glucocorticoid receptor. For this procedure, the GR first undergoes a major purification by anion exchange chromatography following hormone binding and on-column receptor transformation. Second, the GR is resolved to homogeneity utilizing a hydrophobic interaction chromatography step which consists of a 2.5 M to 0 M NaCl gradient elution of contaminating proteins followed by displacement of GR by CHAPS. GR at both stages of purification was able to activate transcription from the glucocorticoid response element containing the promoter region of the long terminal repeat of the mouse mammary tumor virus. This simple and efficient methodology should be of a considerable advantage for studies of the biology of the active, full-length GR.

Role of the TATA box in transcription of the mouse mammary tumor virus long terminal repeat.

An in vitro transcription system from mammary cells was established to study transcription of the long terminal repeat (LTR) of the mouse mammary tumor virus (MMTV). Experiments with progressive 5'-deletion constructs of the MMTV LTR revealed that a 19-base pair (bp) region from -41 to -23 bp, encompassing the TATA box and flanking DNA sequence, was as transcriptionally active as larger promoter constructs, both in nuclear extracts from human mammary cell lines (T47D and MCF7) and a nonmammary cell line (HeLa). The cell-free system was capable of supporting transcriptional induction by factors binding upstream of the TATA box, however, since purified glucocorticoid receptor-induced transcription in larger promoter constructs encompassing the MMTV hormone-responsive elements. Transcription from two other promoters, the adenovirus major late promoter and the human immunodeficiency virus LTR, also revealed a significant transcriptional contribution of upstream elements. The 19-bp TATA region from the MMTV LTR was shown to have considerably more activity in this transcription system than comparable TATA regions from other promoters. Sequences critical to the MMTV TATA region were evaluated by single base pair mutagenesis and found to comprise a consensus TATA box sequence, TATAAAA, as well as a single A just upstream of the TATAAAA sequence. Thus, the high level of basal transcription observed with the TATA region from MMTV is due to a perfect consensus TATA box sequence and a single base immediately 5' adjacent. It is likely that the high basal rate of transcription observed with this TATA box region on histone-free templates represents an inappropriate level of basal expression and that a complete evaluation of transactivation mechanisms in this system will require the recapitulation in vitro of the chromatin-mediated repressive state that exists in vivo.

Loss of mouse epidermal protein kinase C isozyme activities following treatment with phorbol ester and non-phorbol ester tumor promoters.

The present study has examined changes in activities and levels of four protein kinase C (PKC) isozymes (PKC alpha, PKC beta, PKC gamma and PKC delta) detectable in mouse epidermal preparations following both single and multiple treatments with 12-O-tetradecanoylphorbol-13-acetate (TPA). In addition, PKC epsilon and PKC eta protein levels were monitored by immunoblotting following TPA application. Finally, PKC isozyme activity profiles were also examined in epidermal preparations from mice treated with single applications of two non-phorbol ester tumor promoters: chrysarobin (CHRY) and okadaic acid (OA). Fifteen minutes following topical treatment with a tumor promoting dose of TPA (3.4 nmol), the activities of PKC beta and PKC gamma decreased in the epidermal cytosol to 30% and 50% of control values, respectively, while these activities were increased in the epidermal particulate fraction by approximately 50%. PKC delta activity, found predominantly in the particulate fraction of control epidermis, was greatly diminished in both subcellular fractions at 15 min while PKC alpha activity was translocated approximately 20% from cytosol to particulate fraction. Significant reductions in all four detectable PKC isozyme activities in both particulate and cytosol fractions were observed 48 h after a single treatment with TPA, although particulate PKC alpha activity appeared to be less affected at this point in time compared to the other PKC isozymes. Immunoblotting analyses of PKC isozyme protein levels after TPA treatment followed the changes in activity for cytosolic PKC alpha, PKC beta and PKC gamma. However, particulate PKC delta and PKC epsilon protein levels remained relatively unchanged while particulate PKC eta protein levels were significantly down-regulated after a single TPA treatment. Multiple topical treatments (twice-weekly for 2 weeks) with TPA produced a pattern of loss followed by only partial recovery of total PKC activity. Furthermore, all four PKC isozyme activities examined by hydroxylapatite (HA) chromatography were significantly reduced, including PKC alpha, after four applications of TPA. Cytosolic PKC alpha, PKC beta and PKC gamma protein levels as determined by immunoblotting again followed the activity profiles; particulate PKC eta protein levels were significantly reduced, whereas particulate PKC delta and PKC epsilon levels again appeared relatively unchanged. Fifteen minutes after topical application of 220 nmol CHRY, an approximately 25% decrease in particulate associated with PKC alpha activity was observed while particulate activities associated with PKC beta, PKC gamma and PKC delta were unaffected by CHRY at this time point.(ABSTRACT TRUNCATED AT 400 WORDS)

Further identification of protein kinase C isozymes in mouse epidermis.

In the current study, the protein kinase C (PKC) isozymes present in mouse epidermis have been identified using immunological and chromatographic methods. Six PKC isozymes, PKC alpha, PKC beta, PKC gamma, PKC delta, PKC epsilon, and PKC zeta, were identified in unfractionated epidermal preparations by protein immunoblotting. The subcellular distribution and presence of these isozymes was further verified by hydroxyapatite (HA) chromatography with the exception of PKE epsilon, which could not be detected following HA chromatography. The five PKC isozymes recovered following HA chromatography were detected in both epidermal cytosol and particulate fractions, although PKC delta was found in a much higher proportion relative to the other PKC isozymes in the particulate fraction using histone H1 as the substrate. The biochemical properties of the epidermal PKC isozymes partially purified by HA chromatography agreed with those reported for other tissues and further supported their immunological identification in epidermal preparations. The activities of HA chromatography peaks corresponding to PKC alpha, PKC beta, and PKC gamma were found to be dependent on both Ca2+ and phosphatidylserine (PtdSer), whereas, the activities of HA peaks corresponding to PKC delta and PKC zeta were Ca(2+)-independent but PtdSer-dependent. The HA peak corresponding to PKC gamma also displayed a characteristic biphasic modulation by arachidonic acid (activation at low, inactivation at high concentrations) and inactivation by preincubation with PtdSer. PKC zeta activity was also characteristic, in that it was dependent on PtdSer and was not increased by the phorbol ester, 12-O-tetradecanoylphorbol 13-acetate. Some differences in substrate specificity were also observed between the epidermal PKC isozymes. The presence of multiple isozymes of PKC in mouse epidermis suggests that the different isozymes may play distinct roles in signal transduction and tumor promotion in this tissue.

O6-alkylguanine-DNA alkyltransferase activity in epidermal tumor and normal epidermal cells of mice of various stocks and strains.

The level of the DNA repair enzyme O6-alkylguanine-DNA alkyltransferase (MGMT) was examined in benign and malignant skin tumors induced with different initiating and promoting agents and from both SENCAR and Sensitive SENCAR Inbred (SSIN) mice. The MGMT levels in the tumors were approximately one-half the level observed in normal surrounding epidermis and in keratinocytes from untreated controls. In addition, a carcinoma-producing cell line, VT 17DT, derived from papillomas in SENCAR mice had no detectable MGMT activity (Mer- phenotype), whereas in the non-tumor forming line, 3PC, MGMT activity was comparable to that in papillomas. The comparatively low level of MGMT in papillomas may contribute to their ease of conversion to squamous cell carcinomas by N-ethyl-N-nitrosourea or n-methyl-N'-nitro-N-nitrosoguanidine. MGMT activity was also determined in the epidermis of non-exposed mice of various stocks and strains. Epidermal MGMT activity was similar to levels in the corresponding livers and was, in general, parallel with stock/strain susceptibility to tumor formation. This is the first report that examined MGMT activity in skin tumors and normal keratinocytes in the mice of several stocks and strains.

Binding of [3H]bryostatin 4 to protein kinase C.

The bryostatins represent a unique class of activators of protein kinase C (PKC) which induce only a subset of the responses typical of the phorbol esters and block those responses to the phorbol esters which they themselves do not induce. To better understand the interaction of the bryostatins with PKC, we have synthesized [26-3H]bryostatin 4 and characterized its binding to PKC. [3H]Bryostatin 4 and [3H]phorbol 12,13-dibutyrate ([3H]PDBu) differed markedly in their binding to PKC reconstituted with phosphatidylserine (PS). The binding affinity of [3H]bryostatin 4 under these conditions was too high to measure and the rate of release of bound bryostatin was much slower than that of the phorbol esters, with a half-time of several hours. These properties caused bryostatin 1 to appear to inhibit [3H]PDBu binding under these conditions in a non-competitive fashion. Both the high potency and the slow rate of release of the bryostatins may contribute to their unique pattern of biological activity. By reconstituting PKC in a mixture of 1.5% Triton X-100:0.3% PS, we were able to establish reversible conditions for [3H]bryostatin 4 binding. Under these latter conditions, binding of [3H]bryostatin 4 was competitively inhibited by PDBu, consistent with both the bryostatin and phorbol esters binding to PKC in a qualitatively similar fashion. Binding affinities to PKC isozymes alpha, beta, and gamma were compared and little difference was found, suggesting that differential recognition by these isozymes does not account for the unique biological activity of the bryostatins.

Potent inhibitory effects of suicide inhibitors of P450 isozymes on 7,12-dimethylbenz[a]anthracene and benzo[a]pyrene initiated skin tumors.

A single dose of 1-ethynylpyrene (EP), 1-vinylpyrene (VP) or 2-ethynylnaphthalene (EN) was applied to the skin of SENCAR mice 5 min before an initiating dose of 7,12-dimethylbenz[a]anthracene (DMBA) or benzo[a]pyrene (B[a]P) and the development of skin tumors then promoted with biweekly topical applications of 12-O-tetradecanoylphorbol-13-acetate (TPA). The application of EP strongly inhibited the formation of skin tumors initiated by either DMBA or B[a]P in a dose-dependent manner. Application of 44 pmol of EP inhibited tumor initiation by 10 nmol of DMBA approximately 25%; application of 440 nmol of EP inhibited tumor initiation by 200 nmol of B[a]P approximately 51%. A high single dose of EP (4.4-44 mumol) nearly eliminated skin tumor initiation by either 10 nmol of DMBA or 200 nmol of B[a]P. Application of VP also inhibited the formation of skin tumors initiated by either DMBA or B[a]P in a dose-dependent manner, but higher doses of VP than of EP were required to produce comparable inhibitions. Application of 44 nmol of VP inhibited tumor initiation by 10 nmol of DMBA approximately 30%; application of 4.4 mumol of VP inhibited tumor initiation by 200 nmol of B[a]P approximately 56%. Application of EN yielded contrasting results. EN inhibited the formation of skin tumors initiated by 10 nmol of DMBA, but the observed dose-dependence was minimal; tumors were decreased about 40% by 3.3 mumol of EN and only about 65% by 132 mumol of EN. A high single dose of EN (132 mumol) increased both the mean number of tumors per mouse and the percentage of mice that developed tumors after initiation by 200 nmol of B[a]P. Topical application of 4.4 mumol of EP, 22 mumol of VP or 33 mumol of EN to the skin of SENCAR mice 5 min before a single initiation dose of 2.5 mumol of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) had a minimal inhibitory effect (14-28%) on the development of skin tumors produced by subsequent biweekly promotion with TPA. A single dose of 44 mumol of EP or 132 mumol of EN followed by biweekly applications of TPA did not produce skin tumors; however, a dose of 44 mumol of VP followed by promotion with TPA produced a low but significant number of skin tumors.(ABSTRACT TRUNCATED AT 400 WORDS)

Partial characterization of epidermal protein kinase C in mice sensitive or resistant to phorbol ester.

The present study has characterized several aspects of the mouse epidermal protein kinase C (PKC) system and compared phorbol ester-sensitive and -resistant mice. Protein immunoblots of partially purified epidermal PKC preparations from SENCAR and C57BL/6 mice indicated the presence of the gamma-, beta-, and alpha-isozymes of PKC in both strains. Hydroxylapatite chromatography profiles of epidermal PKC isozymes from SENCAR and C57BL/6 mice revealed three major peaks of PKC activity eluting in fractions similar to those observed in chromatograms of brain tissue and corresponding to PKC-gamma, -beta, and -alpha. Further analyses of hydroxylapatite chromatography fractions revealed that PKC-gamma and -beta were present in approximately similar proportions and were much more abundant than PKC-alpha. This distribution of epidermal PKC isozymes was similar in both strains. After a single topical application of 3.4 nmol 12-O-tetradecanoylphorbol-13-acetate (TPA) to SENCAR mouse epidermis, total PKC activity in the cytosol fraction decreased rapidly to about 50% of control within 15 min and was accompanied by an increase (approximately 150% of control) of PKC activity in the membrane fraction. At 4 h, PKC activities were significantly lower than the control levels and remained downregulated through 96 h with a maximal decrease (to approximately 25-30% of the control) in both cytosol and membrane fractions at h. PKC activity returned to control levels by 168 h. Ca++/phospholipid-independent kinase activity was the same as control levels at 15 min, 1 h, and 4 h after TPA treatment but was elevated above control levels at 24 h, 48 h, and 96 h, and by 168 h returned essentially to control levels. No differences were found in the magnitude or kinetics of TPA-induced translocation and downregulation of total PKC or appearance of Ca++/phospholipid-independent kinase activity between SENCAR, DBA/2, and C57BL/6 mice. Scatchard analyses using a two binding site model revealed that the apparent Kd and Bmax values for binding of PDBu to epidermal cytosol and membrane fractions were similar between SSln, SENCAR, DBA/2, and C57BL/6 mice. The present results demonstrate for the first time that mouse epidermis contains significant amounts of the three major PKC isozymes that are present in brain, especially PKC-gamma. In addition, topical application of a promoting dose of TPA did not lead to complete loss of PKC activity in either the membrane or cytosol fractions of mouse epidermis. In conclusion, no differences were observed between phorbol ester-sensitive and -resistant mice in any aspect of epidermal PKC examined.

Protein kinase C-gamma is present in adriamycin resistant HL-60 leukemia cells.

The isoform pattern of protein kinase C (PKC) was examined in wild-type and Adriamycin-resistant (HL-60/AR) HL-60 leukemia cells. Analyses were carried out by immunoblotting with mouse monoclonal antibodies against PKC-alpha and PKC-beta and a rabbit polyclonal antibody against the variable (V3) region of PKC-gamma. HL-60/AR cells contained an equivalent level of PKC-alpha and a lower amount of PKC-beta than HL-60 cells. In contrast, only HL-60/AR cells contained PKC-gamma. These results indicate that the regulation of this family of isoenzymes is altered in drug-resistant cells.

Effects of pyrimidine nucleoside phosphorylase inhibitors on hepatic fluoropyrimidine elimination in the rat.

The breakdown of 5-fluoro-2'-deoxyuridine (FdUrd) to 5-fluorouracil (FUra) is catalyzed by the pyrimidine nucleoside phosphorylases, uridine phosphorylase and thymidine phosphorylase. The effects of nucleoside phosphorylase inhibitors on FdUrd and FUra elimination by the isolated perfused rat liver were investigated. The inhibitor was injected into the perfusion reservoir 5 min before FdUrd or FUra, and serial perfusion fluid samples were collected for fluoropyrimidine analysis. The disappearance of each fluoropyrimidine followed Michaelis-Menten kinetics, as shown previously. 6-Benzyl-2-thiouracil, a thymidine phosphorylase-selective inhibitor, and 1-(2'-deoxy-beta-D-glucopyranosyl)thymine, a uridine phosphorylase-selective inhibitor, each decreased the rate of FdUrd disappearance (apparent Ki, 1.4-1.6 and 3.8 mM, respectively) but had no direct effect on FUra disappearance. However, 6-benzyl-2-thiouracil decreased the peak concentration of FUra derived from administered FdUrd and increased the t 1/2 of disappearance of derived FUra due to its delayed formation. 2,6-Dihydroxypyridine, a uridine phosphorylase-selective inhibitor, decreased the rate of FdUrd disappearance (apparent Ki, 12.4-16.2 microM) and directly inhibited FUra elimination (apparent Ki, 4.3-5.3 microM). 2,4-Dihydroxypyridine, which does not inhibit pyrimidine nucleoside phosphorylases, directly inhibited FUra elimination (apparent Ki, 77 microM) and also decreased the rate of FdUrd disappearance, possibly due to product (FUra) inhibition. It was concluded that the hepatic elimination of FdUrd is slowed by pyrimidine nucleoside phosphorylase inhibitors and that some of these drugs block FUra, as well as FdUrd, elimination.