Mark Danielsen

Sequence specific detection of DNA using nicking endonuclease signal amplification (NESA).

We have developed a new method for identifying specific single- or double-stranded DNA sequences called nicking endonuclease signal amplification (NESA). A probe and target DNA anneal to create a restriction site that is recognized by a strand-specific endonuclease that cleaves the probe into two pieces leaving the target DNA intact. The target DNA can then act as a template for fresh probe and the process of hybridization, cleavage and dissociation repeats. Laser-induced fluorescence coupled with capillary electrophoresis was used to measure the probe cleavage products. The reaction is rapid; full cleavage of probe occurs within one minute under ideal conditions. The reaction is specific since it requires complete complementarity between the oligonucleotide and the template at the restriction site and sufficient complementarity overall to allow hybridization. We show that both Bacillus subtilis and B. anthracis genomic DNA can be detected and specifically differentiated from DNA of other Bacillus species. When combined with multiple displacement amplification, detection of a single copy target from less than 30 cfu is possible. This method should be applicable whenever there is a requirement to detect a specific DNA sequence. Other applications include SNP analysis and genotyping. The reaction is inherently simple to multiplex and is amenable to automation.

Differential Regulation of Androgen and Glucocorticoid Receptors by Retinoblastoma Protein

The androgen receptor (AR) plays a major role in the development and maintenance of male primary and secondary sexual characteristics. The growth promoting effects of androgens are clearly seen in prostate cancer where treatment by androgen ablation usually leads to tumor regression, followed sometime later, by growth of tumor cells that are resistant to endocrine therapy. We have found that the level of pRB in cells controls AR activity. Overexpression of pRB leads to increased transcriptional activity of the AR. This is similar to the previously reported potentiation of glucocorticoid receptor activity by pRB. In contrast, loss of pRB activity inhibits AR but not glucocorticoid receptor activity. This inhibition correlates with the unique ability of the AR to form a protein-protein complex with pRBin vitro. The site of interaction with pRB lies within the N-terminal domain of the AR and co-localizes with the region of the AR that specifies a requirement for pRB. Thus, the AR has a novel requirement for pRB raising the possibility that the growth promoting activity of AR is due to its direct interaction with pRB. Furthermore, loss of pRB activity during progression of prostate cancer may directly result in a decreased response to androgens.

Loss of Androgen Receptor Transcriptional Activity at the G1/S Transition

Androgens are essential for the differentiation, growth, and maintenance of male-specific organs. The effects of androgens in cells are mediated by the androgen receptor (AR), a member of the nuclear receptor superfamily of transcription factors. Recently, transient transfection studies have shown that overexpression of cell cycle regulatory proteins affects the transcriptional activity of the AR. In this report, we characterize the transcriptional activity of endogenous AR through the cell cycle. We demonstrate that in G0, AR enhances transcription from an integrated steroid-responsive mouse mammary tumor virus promoter and also from an integrated androgen-specific probasin promoter. This activity is strongly reduced or abolished at the G1/S boundary. In S phase, the receptor regains activity, indicating that there is a transient regulatory event that inactivates the AR at the G1/S transition. This regulation is specific for the AR, since the related glucocorticoid receptor is transcriptionally active at the G1/S boundary. Not all of the effects of androgens are blocked, however, since androgens retain the ability to increase AR protein levels. The transcriptional inactivity of the AR at the G1/S junction coincides with a decrease in AR protein level, although activity can be partly rescued without an increase in receptor. Inhibition of histone deacetylases brings about this partial restoration of AR activity at the G1/S boundary, demonstrating the involvement of acetylation pathways in the cell cycle regulation of AR transcriptional activity. Finally, a model is proposed that explains the inactivity of the AR at the G1/S transition by integrating receptor levels, the action of cell cycle regulators, and the contribution of histone acetyltransferase-containing coactivators.

Methoxyacetic Acid Disregulation of Androgen Receptor and Androgen-Binding Protein Expression in Adult Rat Testis

Abstract Chemical agents can disrupt the balance between survival and apoptosis during spermatogenesis and thus give rise to reduced counts of spermatozoa (oligospermia). One such agent that produces significant germ cell apoptosis at specific stages of the cycle of the seminiferous epithelium is methoxy acetic acid (MAA), the active metabolite of a commonly used solvent, methoxyethanol. Although MAA gives rise to apoptosis of pachytene spermatocytes, it is not known whether MAA exerts a direct effect on germ cells or whether it also affects other testicular cell types such as the Sertoli cells. In the present investigation, we tested the hypothesis that MAA has direct effects on Sertoli cells in vivo. In MAA-treated rats, stage-specific expression of androgen receptor (AR) protein in Sertoli cells was significantly altered, as determined by AR immunohistochemistry. In MAA-treated animals, high AR expression was found in Sertoli cells coincident with the MAA-induced apoptosis of late-stage pachytene spermatocytes. The altered expression of AR in MAA-treated animals was also seen in seminiferous tubules harvested by laser capture microdissection. In addition to effects on AR expression, androgen-binding protein (ABP) mRNA levels were also altered in a stage-specific manner. Using a different system for mouse Sertoli cell lines TM4 and MSC-1, positive for either AR or ABP, respectively, we found a direct effect of MAA on ABP protein and mRNA expression in the MSC-1 cell but did not detect an effect on AR protein or mRNA expression in TM4 cells. Mouse fibroblasts that express endogenous AR were stably transfected with two AR promoter/reporter systems (MMTV-CAT and probasin-luciferase, respectively). We used these fibroblasts to examine the ability of MAA to potentiate dihydrotestosterone (DHT) activation of AR. Although MAA did not activate AR directly, it did potentiate DHT activation of the AR by 2- to 4-fold. MAA altered the expression level of AR and ABP in vivo and increased AR transcriptional activity in tissue culture cells. The abnormal spermatogenesis generated by MAA is at least partly due to direct effects on Sertoli cells. It is still unclear whether MAA elicits a proapoptotic signal from Sertoli cells or diminishes a prosurvival signal required by germ cells downstream to altering AR and ABP expression in a stage-specific fashion.

A Stu I polymorphism in the human androgen receptor gene (AR)

Fig. 1. Autoradiograph of a Stu I polymorphism in human DNA samples probed with a 542 bp Pvu I1 hAR cDNA fragment (bp 1040 to 1582). The absence of a Stu I site yields a 1.3 kb band. This 1.3 kb band is cut into 0.85 kb and 0.45 kb bands when the Stu I site is present but the 0.45 kb band is not detected with this probe. Lane 1, hemizygote for the 1.3 kb allele; lane 2 , hemizygote for the 0.85 kb allele; lane 3, heterozygote (HeLa cell DNA).

Selective effects of 8-Br-cAMP on agonists and antagonists of the glucocorticoid receptor

RU486 has been reported to be a glucocorticoid receptor (GR) and a progesterone receptor (PR) antagonist. We have analysed RU486 activity on the GR in WCL-2 (CHO) cells and in COS-7 cells transiently transfected with the mouse GR and with the reporter MMTVCAT (MCAT). These cell lines do not contain any active progesterone or androgen receptors. In both cell lines RU486 is a partial agonist of the GR with 10–15% of the activity of dexamethasone. As expected, RU486 is also a partial antagonist of the GR. Treatment of COS-7 cells with 8-Br-cAMP increases the agonist activity of both dexamethasone and RU486. This cAMP induced superactivation is seen with all steroids that have full or partial agonist activity. In contrast, the activities of ZK98.299 and R5020, which are complete antagonists of the GR without any agonist activity, are not affected by 8-Br-cAMP treatment. This effect of 8-Br-cAMP is not seen in WCL2 cells. 8-Br-cAMP, therefore, is not a switch which changes antagonists to agonists but is, rather, a cell specific activator of all agonists whether they have full or only partial agonist activity.

Characterization of ricin toxin family members from Ricinus communis.

Ricin inhibits translation by removal of a specific adenine from 28S RNA. The Ricinus communis genome encodes seven full-length ricin family members. All encoded proteins have the ability of hydrolyzing adenine in 28S rRNA. As expected, these proteins also inhibited an in vitro transcription/translation system. These data show that the ricin gene family contains at least seven members that have the ability to inhibit translation and that may contribute to the toxicity of R. communis.

The Nuclear Receptor Resource Project

We have expanded the original Glucocorticoid Receptor Resource (GRR) database to include several individual resources as part of a larger project called the Nuclear Receptor Resource (NRR). In addition to the GRR, the NRR currently features the Thyroid Hormone Receptor Resource, the Androgen Receptor Resource, the Mineralocorticoid Receptor Resource, the Vitamin D Receptor Resource, and the Steroid Receptor Associated Proteins Resource. The goal of the NRR project is to provide a comprehensive resource for information on the nuclear receptor superfamily, and to provide a forum for the dissemination and discussion of both published and unpublished material on these proteins. Although the individual resources are managed from different servers, all the files are integrated and can be accessed through the projects Home Page, housed at http://nrr. georgetown.edu/nrr.html. In the near future, we hope to expand the project to contain information on other nuclear receptors and to better our electronic publication system. To accomplish this, we encourage the involvement of nuclear receptor investigators in the NRR.

8-Br-cAMP Does Not Convert Antagonists of the Glucocorticoid Receptor into Agonists

Publisher Summary The mouse glucocorticoid receptor (GR) is a member of the steroid and thyroid hormone receptor superfamily. A subgroup of this family includes the glucocorticoid, progesterone (PR), androgen (AR), and mineralocorticoid (MR) receptors. This chapter presents a study to characterize the GR response to various steroids in the presence and absence of 8-Br-cAMP. 8-Br-cAMP increases the activity of both full and partial agonists but has no effect on the complete antagonists R5020 and ZK98.299. The studies explained in this chapter suggest that 8-Br-cAMP acts as a switch converting RU486 from an antagonist of the GR into a partial agonist. This is not the case in either CHO cells or COS-7 cells, as in both cell lines RU486 induces MCAT even in the absence of 8-Br-cAMP. Treatment of COS-7 cells with 8-Br-cAMP increases the agonist activity of RU486 and other partial agonists such as progesterone and to a lesser extent full agonists. R5020 and ZK98.299 bind to the GR but do not activate transcription even in the presence of 8-Br-cAMP. Therefore, instead of converting an antagonist to an agonist, 8-Br-cAMP increases the activity of all agonists. ZK98.299 binds tightly to the GR but has no transcriptional activity. ZK98.299 differs from RU486 in the substituents and conformation of the D-ring. This indicates that the 4-dimethylaminophenyl group at the 11β position of both RU486 and ZK98.299 is not the most critical factor in determining antagonistic activity.

Expression of the Xenopus laevis Mineralocorticoid Receptor during Metamorphosis

Publisher Summary Steroid and thyroid hormone receptors play a major role in differentiation and development. These receptors are composed of three separate domains: (1) a C-terminal hormone-binding domain, (2) a centrally located DNA binding domain, and (3) a N-terminal domain that has been termed a modulatory domain as it is involved in transcription activation. This chapter presents a study to report on the cloning of the X. laevis MR (xMR) and to describe initial experiments on its expression. The receptor is highly homologous to its human and a rat counterpart except that it has an extended DNA-binding domain. The xMR is expressed at a low level throughout metamorphosis, although the mRNA levels do increase as metamorphosis progresses. The chapter presents a comparison of the xMR with the rat and human clones. The xMR DNA-binding domain has four extra amino acids located between the two zinc fingers of the DNA-binding domain, making it the longest DNA-binding domain found in the superfamily. The four extra amino acids in the xMR are the result of the use of a different splice site in Xenopus, and they do not inhibit transcription activity.