Steroid-independent activation of steroid receptors in mammalian and yeast cells and in breast cancer.
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Animals↗
Biomedical subjects
D Picard
Publications and source records attributed to D Picard.
Restorative treatments received by children covered by a universal, publicly financed, dental insurance plan.
OBJECTIVES: This study sought to identify risk markers associated with the provision of new restorations in children and to investigate whether the carious status of a tooth surface is associated with the restorative decisions of dentists. METHODS: A total of 911 schoolchildren in grades one, two, and three were randomly selected from the island of Montreal, Quebec, Canada. Dental examinations were carried out in 1990, 1991, and 1992. Tooth surfaces of first permanent molars were classified as sound, noncavitated, and cavitated. The carious status of a tooth was matched with restorative decisions reported to the insurance board. RESULTS: The presence of a carious cavity was a strong risk marker for placement of new restorations (odds rations > or = 4.11). After one year, less than 2 percent of sound tooth surfaces of first permanent molars were restored and about 21 percent of noncavitated tooth surfaces were restored. When new class I restorations placed in maxillary first permanent molars within 3-6 months after the baseline examination were evaluated, we found that between 73 percent and 86 percent of these new restorations were placed in sound or noncavitated tooth surfaces. A similar trend also was observed in mandibular first permanent molars. Poor agreement between epidemiologic diagnosis and restorative decisions was found. The restorative profile of dentists was a significant risk marker for placement of new restorations. CONCLUSION: The majority of new restorations in first permanent molars were placed in sound and noncavitated tooth surfaces because of the ubiquitous prevalence of these tooth surfaces and the validity problems of current caries diagnosis methods.
Child↗
Functional analysis of the yeast 40 kDa cyclophilin Cyp40 and its role for viability and steroid receptor regulation.
We have identified and characterized a homolog of the 40 kDa cyclophilins in the budding yeast Saccharomyces cerevisiae. At the amino acid level, this novel yeast cyclophilin, termed Cyp40, is 47% identical to human cyclophilin-40. Recombinant Cyp40 produced in bacteria has a peptidyl-prolyl cis-trans isomerase activity with a catalytic efficiency (k[cat]/K[m]) of 0.5 x 10(6)M(-1)s(-1), which can be inhibited by cyclosporin A with an IC50 value of 60nM. Using a polyclonal antibody against Cyp40 we have found that Cyp40 is predominantly cytoplasmic, and that its expression is induced 3-4-fold by heat shock. Moreover, Cyp40 can be coprecipitated from yeast extracts with the cytosolic molecular chaperone Hsp90. Surprisingly, a Cyp40-deficient yeast strain is fully viable at normal and elevated temperatures. Cyp40 is also dispensable for normal regulation of vertebrate steroid receptors in yeast. While other immunophilins could conceivably compensate a Cyp40 defect, our results are compatible with the notion that immunophilins may be fortuitous partners in the biochemically established steroid receptor-Hsp90 complex.
Amino Acid Isomerases↗
School-related issues among HIV-infected children.
OBJECTIVE: Many children with human immunodeficiency virus (HIV) infection are surviving long enough to reach school age. This study describes issues related to school attendance and disclosure of HIV infection in a population of HIV-infected children. METHODS: A statewide pediatric HIV surveillance system was used to collect data on school-age (>/=5 years old) HIV-infected children. In addition, HIV clinic nurses familiar with the child's history participated in a cross-sectional survey that collected information on school-related issues during the 1993-1994 school year. RESULTS: Of the 92 school-age children, only 3 were too ill to attend school. Another 5 children were home-schooled. Of the 84 who attended school outside the home, 25% had severe symptoms of HIV infection (Centers for Disease Control and Prevention [CDC] clinical category C). Absence from school ranged from less than 2 weeks during the year for half of the children (51%) to more than 8 weeks for 9 children (12%). Twenty-nine percent of the children received medication in school, usually administered by the school nurse. Over two thirds of the 50 children ages 5 to 10 years had not been told that they had HIV infection. Only 1 of the 20 children more than 10 years of age was not aware of her HIV infection. For 53% of the children attending school, no school personnel had been informed of the child's HIV infection. Administration of HIV medications at school, age of child, and treatment at one particular HIV clinic were associated with the parents' decision to inform school personnel. In the 47% of cases where the school had been informed, school nurses were most frequently notified, followed by principals and teachers. CONCLUSION: Only 3% of school-age children were too ill to attend school, and almost all were enrolled in public schools. The number of HIV-infected children reaching school age will continue to grow, and public schools will bear the responsibility for educating these children. Health care providers will increasingly be called upon for guidance by both educators and families to assure that HIV-infected children receive the best education possible.
Absenteeism↗
Methodological validation and clinical usefulness of carbon-14-urea breath test for documentation of presence and eradication of Helicobacter pylori infection.
UNLABELLED: A simple [14C]urea breath test (C-14-UBT) was validated with aims of determining accuracy in documenting both the presence and proof of eradication of Helicobacter pylori infection. METHODS: Fifty-six dyspeptic patients had endoscopy with biopsies and C-14-UBT. Eleven biopsy-proven H. pylori-negative patients allowed C-14-UBT normal value determination. Forty-three patients with recurrent peptic ulcer disease and biopsy-proven H. pylori infection were included in an antimicrobial eradication protocol. Endoscopy with biopsies and C-14-UBT were done again 8 wk after initiation of treatment in 35 patients. For C-14-UBT, 185 kBq (5 microCi) of [14C]urea was swallowed. Breath samples obtained up to 20 min were counted to calculate AS20, [(% 14CO2 dose excreted/mmol of CO2) x kg] at 20 min. Combined histologic and microbiologic analyses of antral biopsies were used as a gold standard. RESULTS: The positivity value was set as AS20 > 0.33% (mean + 3 s.d. of AS20 in H. pylori-negative patients). Diagnosis of H. pylori infection was correct with C-14-UBT in 55/56 patients (44 true-positive, 11 true-negative and 1 false-negative; sensitivity = 98%; specificity = 100%). As a proof of eradication, C-14-UBT correctly classified 33/35 patients (5 true-positive, 28 true-negative and 2 false-positive; sensitivity = 100%; specificity = 93%). The C-14-UBT global performance yielded sensitivity, specificity and accuracy of 98%, 95% and 97%, respectively. A significant correlation (r = 0.84) was found between AS20 and the number of H. pylori colonies on culture. CONCLUSION: This C-14-UBT is highly accurate both for diagnosis and proof of eradication of H. pylori infection and reflects the antral bacterial load. It is simple, fast and inexpensive, and it is therefore suitable for clinical practice.
Breath Tests↗
Two eukaryote-specific regions of Hsp82 are dispensable for its viability and signal transduction functions in yeast.
The 90-kDa heat shock protein (Hsp90) is a molecular chaperone that is very abundant even at normal temperature. It is highly conserved and essential for viability in yeast. To delineate functional domains of Hsp90, we have performed a deletion analysis of one of the two Hsp90 isoforms from budding yeast, Hsp82. The Hsp82 derivatives were tested for complementation of a Hsp90-deficient yeast strain and for their ability to function in two signal transduction pathways that depend on Hsp90. Surprisingly, we found that two salient features of Hsp90 sequences from eukaryotes, the N-terminal charged domain and the extremely conserved C-terminal pentapeptide MEEVD, are dispensable for viability as well as for proper regulation of vertebrate steroid receptors and for pheromone signaling. Moreover, we describe, to our knowledge, the first dominant negative mutant of Hsp90; A Hsp82 derivative that lacks amino acids 538-552 fails to complement but has a dominant negative effect on viability of wild-type strains at moderately elevated temperatures. This mutant may become a valuable tool to study Hsp90 functions both in yeast and in mammalian cells.
Amino Acid Sequence↗
Growth inhibition by Abl requires an interplay of its SH2 and tyrosine kinase domains.
Overexpression of c-Abl tyrosine kinase can be growth inhibitory in certain fibroblast cell lines. Using a series of conditional chimeras between Abl and Src, we have now further dissected the Abl protein to determine which domains are required for this function. We found that growth inhibition, unlike transformation by oncogenic forms of Abl, is dependent on the presence of the cognate SH2 and tyrosine kinase domains. Since growth inhibition correlates with low tyrosine kinase activity, it may involve highly specific interactions of target proteins with both domains without the processivity of phosphorylation associated with oncogenic Abl.
3T3 Cells↗
Activation of the unliganded estrogen receptor by EGF involves the MAP kinase pathway and direct phosphorylation.
The estrogen receptor (ER) can be activated as a transcription factor either by binding of cognate estrogenic ligand or, indirectly, by a variety of other extracellular signals. As a first step towards elucidating the mechanism of 'steroid-independent activation' of the ER by the epidermal growth factor (EGF), we have mapped the ER target domain and determined the signaling pathway. We show that the N-terminal transcriptional activation function AF-1, but not the C-terminal AF-2, is necessary for the EGF response. Both the EGF-induced hyperphosphorylation and the transcriptional activation of the unliganded ER depend on a phosphorylatable serine residue at position 118. However, its phosphorylation is not sufficient and, hence, there must be other target domains or proteins which fulfill an additional requirement for EGF signaling through the ER. Using dominant-negative Ras and MAP kinase kinase (MAPK kinase) and constitutively active MAPK kinase mutants, we show that EGF activates the ER by signaling through the MAPK pathway suggesting that MAPK directly phosphorylates the critical serine 118. Our results also imply that the steroid-independent activation of a variety of ER mutants, which arise during the malignant progression of breast tumors, may contribute to tamoxifen resistance.
Epidermal Growth Factor↗
Heat shock protein hsp90 regulates dioxin receptor function in vivo.
The dioxin (aryl hydrocarbon) receptor is a ligand-dependent basic helix-loop-helix (bHLH) factor that binds to xenobiotic response elements of target promoters upon heterodimerization with the bHLH partner factor Arnt. Here we have replaced the bHLH motif of the dioxin receptor with a heterologous DNA-binding domain to create fusion proteins that mediate ligand-dependent transcriptional enhancement in yeast (Saccharomyces cerevisiae). Previously, our experiments indicated that the ligand-free dioxin receptor is stably associated with the 90-kDa heat shock protein, hsp90. To investigate the role of hsp90 in dioxin signaling we have studied receptor function in a yeast strain where hsp90 expression can be down-regulated to about 5% relative to wild-type levels. At low levels of hsp90, ligand-dependent activation of the chimeric dioxin receptor construct was almost completely inhibited, whereas the activity of a similar chimeric construct containing the structurally related Arnt factor was not affected. Moreover, a chimeric dioxin receptor construct lacking the central ligand- and hsp90-binding region of the receptor showed constitutive transcriptional activity in yeast that was not impaired upon down-regulation of hsp90 expression levels. Thus, these data suggest that hsp90 is a critical determinant of conditional regulation of dioxin receptor function in vivo via the ligand-binding domain.
Animals↗
Cell cycle arrest by tyrosine kinase Abl involves altered early mitogenic response.
Activated forms of the nuclear and cytoplasmic tyrosine kinase c-Abl are completely cytoplasmic and oncogenic. The overexpression of c-Abl, and in certain fibroblast cell lines even of v-Abl, leads to a cell cycle arrest revealing an alternative Abl function. To facilitate the analysis of this growth inhibitory function we have taken advantage of regulable Abl-estrogen receptor (ABL:ER) fusion proteins. Oncogenic in the presence of estrogen, they are reversibly switched to inhibit cell proliferation upon removal of hormone. Using this system, we demonstrate that inhibition is effected by Abl derivatives which we have previously shown to be hypo-phosphorylated and to have low kinase activity. Since an almost exclusively cytoplasmic ABL:ER protein is fully growth inhibitory, relevant interactions may occur in the cytoplasm. We identify the cell cycle arrest as an early G1 or G0-like block. Interestingly, growth inhibition correlates with an altered expression pattern of early serum response genes; c-Jun mRNA and c-Fos protein levels are elevated in Abl-blocked cells. In view of the two functional modes of overexpressed Abl proteins, one can speculate that normal c-Abl may be involved in relaying growth regulatory signals from the membrane to the nucleus.
3T3 Cells↗
Trypanosoma cruzi heat-shock protein 90 can functionally complement yeast.
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Amino Acid Sequence↗
Noninvasive diagnosis of Caroli syndrome associated with congenital hepatic fibrosis using hepatobiliary scintigraphy.
A patient who was studied for portal hypertension had an enlarged liver and multiple hepatic cysts on abdominal CT scan. He underwent hepatobiliary scintigraphy using Tc-99m mebrofenin which documented communication of the cysts with the main biliary tree and allowed a noninvasive diagnosis of Caroli syndrome associated with congenital hepatic fibrosis.
Adult↗
The hormone binding domain of the mineralocorticoid receptor can regulate heterologous activities in cis.
Steroid receptors are maintained inactive in the absence of cognate ligand partly because of repression by their hormone binding domain (HBD). Proteins complexed with the unliganded HBD of vertebrate steroid receptors, including the heat-shock protein 90, have been implicated as components of a molecular switch. As such, the HBDs of both the glucocorticoid and estrogen receptors have been shown to be autonomous regulatory cassettes which can subject heterologous activities resident on the same polypeptide to hormonal control. We show that the HBD of the mineralocorticoid receptor (MR) carries a similar "protein inactivation" function. Thus, the MR HBD can be used as a movable regulatory domain, a powerful tool for aldosterone regulation of chimeric proteins.
Aldosterone↗
Regulation of protein function through expression of chimaeric proteins.
The hormone-binding domain of steroid receptors can be used to subject heterologous protein functions to hormonal control in cis. Recent studies have established that these regulatory domains constitute a set of up to five different reversible molecular switches for the post-translational regulation of a wide variety of cytoplasmic and nuclear proteins, including kinases. This approach has been applied both in vertebrate cells and in budding yeast.
Animals↗
Regulation of protein activities by fusion to steroid binding domains.
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Adenoviridae↗
Modulation of cyclin gene expression by adenovirus E1A in a cell line with E1A-dependent conditional proliferation.
To investigate how adenovirus E1A controls cell proliferation, we have fused E1A to the hormone-binding domain of the human estrogen receptor (ER) and introduced the E1A-ER chimeric gene together with an activated ras gene into primary rat embryo fibroblasts. Cell lines derived from this transfection proliferate in an estrogen-dependent manner. Estrogen-dependent activation of E1A-ER led to a rapid induction of both cyclin E and cyclin A gene expression. In contrast, levels of cyclin D1 were strongly reduced by activation of E1A-ER. Similar changes in cyclin gene expression were observed when primary human fibroblasts were infected with wild-type adenovirus and when adenovirus E1A was stably expressed in NIH 3T3 cells. Our findings suggest that activation of cyclin A and E, but not D1, gene expression by E1A precedes and may be responsible for E1A-dependent cell proliferation. In contrast, we found that quantitative disruption of complexes between the E2F transcription factor and the retinoblastoma protein is not required for E1A-dependent S-phase entry.
Adenovirus E1A Proteins↗
Fusion of GAL4-VP16 to a steroid-binding domain provides a tool for gratuitous induction of galactose-responsive genes in yeast.
We demonstrate that the hormone-binding domain (HBD) of the human estrogen receptor (ER) can function as an autonomous regulatory domain in the budding yeast, Saccharomyces cerevisiae. As in mammalian cells, the HBD can subject the activity of a heterologous protein, which is fused to it, to hormonal control. Thus, a chimeric transcriptional activator consisting of (i) the DNA-binding domain of GAL4, (ii) the ER HBD, and (iii) the activation domain of viral protein 16 (VP16) stimulates both episomal and integrated reporter genes exclusively in the presence of steroid hormone. Steroids being gratuitous signals for yeast, this fusion protein is a convenient tool for highly regulated production of proteins of interest. Notably, it can be exploited to activate the commonly used galactose-inducible expression vectors without switching the carbon source.
DNA, Recombinant↗
Evidence that the hormone binding domain of steroid receptors confers hormonal control on chimeric proteins by determining their hormone-regulated binding to heat-shock protein 90.
Previously, it has been shown that the hormone binding domain of the glucocorticoid receptor acts as a transferable regulatory cassette that can confer hormonal control onto chimeric proteins [Picard, D., Salser, S. J., & Yamamoto, K. R. (1988) Cell 54, 1073-1080]. The hormone binding domain of the glucocorticoid receptor contains its site of interaction with the 90-kDa heat-shock protein, hsp90 [Dalman, F. C., Scherrer, L. C., Taylor, L. P., Akil, H., & Pratt, W. B. (1991) J. Biol. Chem. 266, 3482-3490]. We have now transfected COS cells with cDNAs for fusion proteins containing beta-galactosidase and portions of the glucocorticoid receptor, and we demonstrate a correlation between hormone regulation of fusion protein localization and binding of the fusion proteins to hsp90. The hormone binding domain (residues 540-795) of the rat glucocorticoid receptor is sufficient for conferring hormone regulation onto a fusion protein and for intracellular binding of a fusion protein to hsp90. The hormone binding domain of the rat glucocorticoid or the human estrogen receptor is also sufficient to permit reticulocyte lysate-mediated refolding of a fusion protein into association with hsp90. Consistent with the results of fusion protein localization in intact cells, binding of a fusion protein to hsp90 blocks binding of antibody directed against the NL1 nuclear localization signal of the glucocorticoid receptor. These observations argue strongly that the hormone binding domain of the glucocorticoid receptor confers hormonal control of fusion proteins by conferring hormone-regulated binding to hsp90.
Animals↗