Biomedical subjects

Jorma J Palvimo

Publications and source records attributed to Jorma J Palvimo.

42 records · Page 3Linked to original sources

PubMed2002

Pattern of somatic androgen receptor gene mutations in patients with hormone-refractory prostate cancer.

Progression to hormone-refractory growth of prostate cancer has been suggested to be mediated by androgen receptor (AR) gene alterations. We analyzed AR for mutations and amplifications in 21 locally recurrent prostate carcinomas treated with orchiectomy, estrogens, or a combination of orchiectomy and estramustine phosphate using fluorescence in situ hybridization, single-strand conformation polymorphism, and DNA sequence analyses. Amplification was observed in 4 of 16 (25%) and amino acid changing mutations was observed in 7 of 21 (33%) of the tumors, respectively. Two (50%) tumors with AR amplification also had missense mutation of the gene. Four of five (80%) cancers that were treated with a combination of orchiectomy and estramustine phosphate had a mutation clustered at codons 514 to 533 in the N-terminal domain of AR. In functional studies, these mutations did not render AR more sensitive to testosterone, dihydrotestosterone, androstenedione, or beta-estradiol. Tumors treated by orchiectomy had mutations predominantly in the ligand-binding domain. In summary, we found molecular alterations of AR in more than half of the prostate carcinomas that recurred locally. Some tumors developed both aberrations, possibly enhancing the cancer cell to respond efficiently to low levels of androgens. Furthermore, localization of point mutations in AR seems to be influenced by the type of treatment.

Estramustine↗

PubMed2002

Androgen receptor acetylation governs trans activation and MEKK1-induced apoptosis without affecting in vitro sumoylation and trans-repression function.

The androgen receptor (AR) is a nuclear hormone receptor superfamily member that conveys both trans repression and ligand-dependent trans-activation function. Activation of the AR by dihydrotestosterone (DHT) regulates diverse physiological functions including secondary sexual differentiation in the male and the induction of apoptosis by the JNK kinase, MEKK1. The AR is posttranslationally modified on lysine residues by acetylation and sumoylation. The histone acetylases p300 and P/CAF directly acetylate the AR in vitro at a conserved KLKK motif. To determine the functional properties governed by AR acetylation, point mutations of the KLKK motif that abrogated acetylation were engineered and examined in vitro and in vivo. The AR acetylation site point mutants showed wild-type trans repression of NF-kappa B, AP-1, and Sp1 activity; wild-type sumoylation in vitro; wild-type ligand binding; and ligand-induced conformational changes. However, acetylation-deficient AR mutants were selectively defective in DHT-induced trans activation of androgen-responsive reporter genes and coactivation by SRC1, Ubc9, TIP60, and p300. The AR acetylation site mutant showed 10-fold increased binding of the N-CoR corepressor compared with the AR wild type in the presence of ligand. Furthermore, histone deacetylase 1 (HDAC1) bound the AR both in vivo and in cultured cells and HDAC1 binding to the AR was disengaged in a DHT-dependent manner. MEKK1 induced AR-dependent apoptosis in prostate cancer cells. The AR acetylation mutant was defective in MEKK1-induced apoptosis, suggesting that the conserved AR acetylation site contributes to a pathway governing prostate cancer cellular survival. As AR lysine residue mutations that abrogate acetylation correlate with enhanced binding of the N-CoR repressor in cultured cells, the conserved AR motif may directly or indirectly regulate ligand-dependent corepressor disengagement and, thereby, ligand-dependent trans activation.

Acetylation↗

PubMed2002

PIAS proteins modulate transcription factors by functioning as SUMO-1 ligases.

PIAS (protein inhibitor of activated STAT) proteins interact with and modulate the activities of various transcription factors. In this work, we demonstrate that PIAS proteins xalpha, xbeta, 1, and 3 interact with the small ubiquitin-related modifier SUMO-1 and its E2 conjugase, Ubc9, and that PIAS proteins themselves are covalently modified by SUMO-1 (sumoylated). PIAS proteins also tether other sumoylated proteins in a noncovalent fashion. Furthermore, recombinant PIASxalpha enhances Ubc9-mediated sumoylation of the androgen receptor and c-Jun in vitro. Importantly, PIAS proteins differ in their abilities to promote sumoylation in intact cells. The ability to stimulate protein sumoylation and the interaction with sumoylated proteins are dependent on the conserved PIAS RING finger-like domain. These functions are linked to the activity of PIASxalpha on androgen receptor-dependent transcription. Collectively, our results imply that PIAS proteins function as SUMO-1-tethering proteins and zinc finger-dependent E3 SUMO protein ligases, and these properties are likely to explain their ability to modulate the activities of various transcription factors.

Animals↗

PubMed2002

Transactivation assay for determination of glucocorticoid bioactivity in human serum.

We have developed a mammalian cell (COS-1) bioassay, which measures glucocorticoid bioactivity (GBA) directly from a small amount of human serum. The assay is based on the expression of human glucocorticoid receptor (GR) together with a coactivator protein and reporter plasmid containing GR response elements upstream of the luciferase gene. Ten microliters of human serum, in duplicate, are added directly to the cell culture medium, and GBA is derived from reporter gene activity. The assay differentiates between biopotencies of synthetic steroids, and importantly, mifepristone (RU486) is able to block glucocorticoid-induced response. The assay is sensitive (<15.6 nM cortisol in fetal calf serum) and precise, with the within- and between-assay coefficients of variation less than 8% and 10%, respectively. We measured serum GBA (bioassay) and cortisol (RIA) levels in 34 asthmatic children (age range, 5.7-14.2 yr) at baseline and after treatment with either inhaled budesonide (800 microg/d, n = 14), fluticasone propionate (500 microg/d, n = 14), or cromones (control group, n = 6). Pretreatment serum GBA and cortisol levels correlated strongly (r = 0.90, P < 0.0001, n = 34). Two months of treatment with inhaled budesonide resulted in excess GBA in circulation, which was not attributable to endogenous cortisol (P < 0.001). In the fluticasone propionate group, the presence of serum excess GBA was at the borderline of statistical significance (P < 0.08) after 2 months of inhalation therapy, and no excess GBA was detected in the cromone group. In conclusion, our bioassay enables measurement of mammalian cell response to bioactive glucocorticoids in circulation and provides a novel means to investigate patients receiving drugs acting through the GR.

Administration, Inhalation↗

PubMed2002

Inhibition of androgen receptor (AR) function by the reproductive orphan nuclear receptor DAX-1.

DAX-1 (NROB1) is an atypical member of the nuclear receptor family that is predominantly expressed in mammalian reproductive tissues. While a receptor function of DAX-1 remains enigmatic, previous work has indicated that DAX-1 inhibits the activity of the orphan receptor steroidogenic factor 1 and the estrogen receptors (ERs), presumably via direct occupation of the coactivator-binding surface and subsequent recruitment of additional corepressors. In vivo evidence points at a particular role of DAX-1 for the development and maintenance of male reproductive functions. In this study, we have identified the androgen receptor (AR) NR3C4 as a novel target for DAX-1. We show that DAX-1 potently inhibits ligand-dependent transcriptional activation as well as the interaction between the N- and C-terminal activation domains of AR. We provide evidence for direct interactions of the two receptors that involve the N-terminal repeat domain of DAX-1 and the C-terminal ligand-binding and activation domain of AR. Moreover, DAX-1, known to shuttle between the cytoplasm and the nucleus, is capable of relocalizing AR in both cellular compartments, suggesting that intracellular tethering is associated with DAX-1 inhibition. These results implicate novel inhibitory mechanisms of DAX-1 action with particular relevance for the modulation of androgen-dependent gene transcription in the male reproductive system.

Androgen Receptor Antagonists↗

PubMed2001

Cooperative coactivation of estrogen receptor alpha in ZR-75 human breast cancer cells by SNURF and TATA-binding protein.

SNURF is a small RING finger protein that binds the zinc finger region of steroid hormone receptors and enhances Sp1- and androgen receptor-mediated transcription in COS and CV-1 cells. In this study, we show that SNURF coactivates both wild-type estrogen receptor alpha (ERalpha) (4-fold)- and HE19 (ERalpha deletion of activation function 1 (AF1)) (210-fold)-mediated activation of an estrogen-responsive element promoter in ZR-75 cells. In mammalian two-hybrid assays in ZR-75 cells SNURF interactions were estrogen (E2)-dependent and were not observed with the antiestrogen ICI 182,780. ERalpha interacted with multiple regions of SNURF; SNURF interactions with ERalpha were dependent on AF2, and D538N, E542Q, and D545N mutations in helix 12 abrogated both SNURF-ERalpha binding and coactivation. Moreover, peptide fusion proteins that inhibit interactions between helix 12 of ERalpha with LXXLL box-containing proteins also blocked ERalpha coactivation by SNURF. However, cotransfection of SNURF with prototypical steroid receptor coactivators 1, 2, and 3 that contain LXXLL box motifs did not enhance E2 responsiveness, whereas TATA-binding protein (TBP) and SNURF cooperatively coactivated ERalpha-mediated transactivation. The results are consistent with a unique model for cooperative coactivation of ERalpha that requires ligand binding, repositioning of helix 12, recruitment of TBP, and interaction with SNURF, which binds both ERalpha and TBP.

Blotting, Western↗