Direct regulation of microRNA biogenesis and expression by estrogen receptor beta in hormone-responsive breast cancer.

Estrogen effects on mammary epithelial and breast cancer (BC) cells are mediated by the nuclear receptors ERα and ERß, transcription factors that display functional antagonism with each other, with ERß acting as oncosuppressor and interfering with the effects of ERα on cell proliferation, tumor promotion and progression. Indeed, hormone-responsive, ERα+ BC cells often lack ERß, which when present associates with a less aggressive clinical phenotype of the disease. Recent evidences point to a significant role of microRNAs (miRNAs) in BC, where specific miRNA expression profiles associate with distinct clinical and biological phenotypes of the lesion. Considering the possibility that ERß might influence BC cell behavior via miRNAs, we compared miRNome expression in ERß+ vs ERß- hormone-responsive BC cells and found a widespread effect of this ER subtype on the expression pattern of these non-coding RNAs. More importantly, the expression pattern of 67 miRNAs, including 10 regulated by ERß in BC cells, clearly distinguishes ERß+, node-negative, from ERß-, metastatic, mammary tumors. Molecular dissection of miRNA biogenesis revealed multiple mechanisms for direct regulation of this process by ERß+ in BC cell nuclei. In particular, ERß downregulates miR-30a by binding to two specific sites proximal to the gene and thereby inhibiting pri-miR synthesis. On the other hand, the receptor promotes miR-23b, -27b and 24-1 accumulation in the cell by binding in close proximity of the corresponding gene cluster and preventing in situ the inhibitory effects of ERα on pri-miR maturation by the p68/DDX5-Drosha microprocessor complex. These results indicate that cell autonomous regulation of miRNA expression is part of the mechanism of action of ERß in BC cells and could contribute to establishment or maintenance of a less aggressive tumor phenotype mediated by this nuclear receptor.

Improving Indigenous patients' access to mainstream health services: the Inala experience.

In 1994, only 12 Indigenous people attended the mainstream general practice in Inala, south-western Brisbane, Queensland. An Indigenous community focus group and telephone interviews revealed deficits such as: few items (eg, artwork) that Indigenous people could identify with; lack of Indigenous staff; staff perceived as unfriendly; inflexibility regarding time; and intolerance of Indigenous children's behaviour. Access to the Inala Indigenous Health Service by Indigenous people improved when these issues were addressed, and has grown significantly every year from 1995 to 2008. Other important factors in improving access include: energetic Indigenous leadership; enabling bulk billing to increase funding; moving to a stand-alone clinic; and engaging with teaching, research and community programs. A Centre of Excellence in Indigenous Primary Health Care is envisaged as the next innovation required to improve access and quality of service, and to close the gap between Indigenous and non-Indigenous health outcomes.

Internet-based home monitoring and education of children with asthma is comparable to ideal office-based care: results of a 1-year asthma in-home monitoring trial.

OBJECTIVE: The goal was to determine whether home asthma telemonitoring with store-and-forward technology improved outcomes, compared with in-person, office-based visits. METHODS: A total of 120 patients, 6 to 17 years of age, with persistent asthma were assigned randomly to the office-based or virtual group. The 2 groups followed the same ambulatory clinical pathway for 12 months. Office-based group patients received traditional in-person education and case management. Virtual group patients received computers, Internet connections, and in-home, Internet-based case management and received education through the study Web site. Disease control outcome measures included quality of life, utilization of services, and symptom control. RESULTS: A total of 120 volunteers (45 female) were enrolled. The groups were clinically comparable (office-based: 22 female/38 male; mean age: 9.0 +/- 3.0 years; virtual: 23 female/37 male; mean age: 10.2 +/- 3.1 years). Virtual patients had higher metered-dose inhaler with valved holding chamber technique scores than did the office-based group at 52 weeks (94% vs 89%), had greater adherence to daily asthma symptom diary submission (35.4% vs 20.8%), had less participant time (636 vs 713 patient-months), and were older. Caregivers in both groups perceived an increase in quality of life and an increase in asthma knowledge scores from baseline. There were no other differences in therapeutic or disease control outcome measures. CONCLUSIONS: Virtual group patients achieved excellent asthma therapeutic and disease control outcomes. Compared with those who received standardized office-based care, they were more adherent to diary submission and had better inhaler scores at 52 weeks. Store-and-forward telemedicine technology and case management provide additional tools to assist in the management of children with persistent asthma.

Concurrent use of metered-dose and dry powder inhalers by children with persistent asthma does not adversely affect spacer/inhaler technique.

BACKGROUND: Studies conducted in adults have suggested that patients who use a metered-dose inhaler/holding chamber spacer (MDI/S) and dry powder inhaler (DPI) concurrently will have poorer MDI/S technique than that of patients who use MDI/S exclusively. To our knowledge, as of August 31, 2006, no studies have been performed in pediatric patients. OBJECTIVE: To compare MDI/S technique scores of children using only MDI/S with scores of those using both MDI/S and DPIs. METHODS: The MDI/S technique of children aged 6-17 years, with persistent asthma, recruited from a general pediatric practice population for an asthma intervention study project was scored using a standardized checklist. MDI/S scores of children who were being treated with maintenance and rescue medication delivered only by MDI/S were compared with those treated with both MDI/S (rescue) and DPI (maintenance). Scores lower than 70% were considered to be inadequate. RESULTS: A total of 117 patients (73 male, 44 female), aged 9.70 +/- 3.1 years (mean +/- SD), with persistent asthma, participated in the study. There were 83 children (54 male, 29 female, age 9.4 +/- 3.2 y) in the MDI/S only group and 34 (19 male, 15 female, age 10.3 +/- 2.9 y) in the MDI/S + DPI group. In the MDI/S + DPI group, Diskus was the DPI used for 32 patients, and Turbuhaler was used by 2 children. Sixteen patients had severe persistent asthma, 80 had moderate persistent asthma, and 21 had mild persistent asthma as classified by National Heart Lung and Blood Institute guidelines. No difference in sex and age demographics existed; however, there was a difference in the distribution of asthma severity between groups (ie, no patients with mild persistent asthma in the MDI/S + DPI group; p < or = 0.01). Mean score for the MDI/S only group was 86 +/- 17% and, for the MDI/S + DPI group, 90.1 +/- 12% (p = 0.15). More patients in the MDI/S group had inadequate scores (18%) compared with those in the MDI/S + DPI group (3%; p < 0.05). CONCLUSIONS: While DPI and MDI/S techniques are markedly different in several significant ways, concurrent use of these inhalers did not adversely affect MDI/S technique scores of pediatric patients with persistent asthma, compared with those using MDI/S alone. Patients in the MDI/S only group had an inadequate MDI/S score (<70%) more often than did patients in the MDI/S + DPI group.

Differentiation of myeloid cell lines correlates with a selective expression of RIZ protein.

BACKGROUND: The retinoblastoma-interacting zinc-finger gene RIZ is expressed in two forms (RIZ1 and RIZ2) that differ for the presence near the N-terminus of RIZ1 of a conserved domain, defined PR (PRDI-BF1-RIZ homology), homologous to a similar domain present in other proteins recognized as tumor suppressor gene products. The RIZ1 form is usually absent or expressed at low levels in tumor cells, whereas RIZ2 is frequently expressed. We investigated a possible involvement of RIZ1 in differentiation control using a myeloid cell maturation model that is easily modulated by retinoids and other agents. MATERIALS AND METHODS: HL60 or NB4 cell lines or patients' leukemic promyelocytes were treated with all- trans -retinoic acid or other agents to induce differentiation. RIZ gene expression was determined with reverse transcriptase polymerase chain reaction (RT-PCR) and RNase protection assay. Immunocytochemistry was performed to assess variation of the intracellular distribution of RIZ protein on all- trans-retinoic acid treatment. Forced expression of RIZ1 protein was obtained with a recombinant adenovirus containing RIZ1 cDNA. RESULTS: Treatment with retinoic acid induced a selective expression of RIZ1 in HL60 cell line. Retinoic acid effect was maximal at 7 days and correlated to the granulocytic differentiation of cells. A similar effect was obtained in retinoic acid-sensitive NB4 cell line or in patients' leukemic promyelocytes, but not in the retinoic acid-resistant cell line NB4.007/6 or in the U937 cell line. Selective expression of RIZ1 was also induced by 12-O-tetradecanoyl-phorbol-13-acetate in the U937 and HL60 cell lines and by 1,25-dihydroxyvitamin D(3) only in HL60 cells. In HL60 cells, RIZ1 was also induced by activation of a retinoid alpha receptor-independent maturation pathway based on retinoid X receptor agonist and protein kinase A synergism. In addition, retinoic acid produced a redistribution of the antigen within the nucleus in these cells. Forced expression of RIZ1 protein induced growth arrest and death of HL60 cells. CONCLUSIONS: The correlation between the selective expression of RIZ1 induced by retinoic acid, 12-O-tetradecanoyl-phorbol-13-acetate, or 1,25-dihydroxyvitamin D(3) and differentiation suggested that RIZ protein was involved in myeloid cell differentiation induced by these agents.

Serum anti-p53 antibodies in lung cancer: comparison with established tumor markers.

As reported earlier, p53 antibodies are detected in the sera of patients with different types of cancer, including lung cancer. In contrast, in the serum of healthy subjects the presence of anti-p53 antibodies is extremely rare. We collected the venous blood samples of 109 patients affected with lung cancer (LC): 57 patients (46 M, 11 F) with non-small-cell carcinoma (NSCLC), 52 others (40 M, 12 F) with small-cell carcinoma (SCLC). Serum p53 antibodies were assayed using ELISA method and all positive sera were confirmed by Western-blot method. In addition, using IRMA methods we assayed serum CEA, TPA, CYFRA21-1 and NSE. Serum p53Ab are detectable (p53Ab-positive) in 35/109 (32.1%) patients with lung cancer. About 17/57 (29.8%) patients affected with NSCLC and 18/52 (34.6%) with SCLC were p53Ab-positive. CEA, TPA, CYFRA21-1 and NSE sensitivity in LC patients (NSCLC+SCLC) is 50.5%, 58.7%, 42.2%, 35.8%, respectively. The lower sensitivity (32.1%) of serum p53Ab is connected with the higher specificity and diagnostic accuracy (100% and 69%, respectively). Out of 35 patients p53Ab-positive, five (14.3%) exhibit only serum p53Ab, while serum values of the established tumor markers were lower than cut-off. Serum p53Ab assessment is a simple and a low-cost assay with a good specificity and diagnostic accuracy that in LC patients can be used at least in association with established tumor markers.

Estradiol induces functional inactivation of p53 by intracellular redistribution.

Estrogen treatment of MCF-7 cells grown in serum-free medium induced a modification of the intracellular distribution of p53 protein. Western blot analysis and immunofluorescence staining showed that p53 was localized in the nucleus of untreated cell and that after 48 h of hormone treatment, it was mostly localized in the cytoplasm. This effect was blocked by the antiestrogen ICI182,780. Intracellular redistribution of p53 was correlated to a reduced expression of the WAF1/CIP1 gene product and to the presence of degradation fragments of p53 in the cytosol. Estradiol treatment prevented the growth inhibition induced by oligonucleotide transfection, simulating DNA damage. This observation indicated that the wild-type p53 gene product present in the MCF-7 cell could be inactivated by estradiol through nuclear exclusion to permit the cyclin-dependent phosphorylation events leading to the G1-S transition. In addition, the estradiol-induced inactivation of p53 could be involved in the tumorigenesis of estrogen-dependent neoplasm.

Tyrosine kinase/p21ras/MAP-kinase pathway activation by estradiol-receptor complex in MCF-7 cells.

The mechanism by which estradiol acts on cell multiplication is still unclear. Under conditions of estradiol-dependent growth, estradiol treatment of human mammary cancer MCF-7 cells triggers rapid and transient activation of the mitogen-activated (MAP) kinases, erk-1 and erk-2, increases the active form of p21ras, tyrosine phosphorylation of Shc and p190 protein and induces association of p190 to p21ras-GAP. Both Shc and p190 are substrates of activated src and once phosphorylated, they interact with other proteins and upregulate p21ras. Estradiol activates the tyrosine kinase/p21ras/MAP-kinase pathway in MCF-7 cells with kinetics which are similar to those of peptide mitogens. It is only after introduction of the human wild-type 67 kDa estradiol receptor cDNA that Cos cells become estradiol-responsive in terms of erk-2 activity. This finding, together with the inhibition by the pure anti-estrogen ICI 182 780 of the stimulatory effect of estradiol on each step of the pathway in MCF-7 cells proves that the classic estradiol receptor is responsible for the transduction pathway activation. Transfection experiments of Cos cells with the estradiol receptor cDNA and in vitro experiments with c-src show that the estradiol receptor activates c-src and this activation requires occupancy of the receptor by hormone. Our experiments suggest that c-src is an initial and integral part of the signaling events mediated by the estradiol receptor.

In vitro phosphorylation and hormone binding activation of the synthetic wild type human estradiol receptor.

A tyrosine kinase purified from calf uterus activates the hormone binding of endogenous estradiol receptor (ER) predephosphorylated and preinactivated by a nuclear phosphotyrosine phosphatase. The kinase also activates and phosphorylates the human estradiol receptor HEO synthesized in vitro, which differs from the wild type receptor HEGO because a glycine is replaced by a valine at position 400. Moreover, the kinase activates and phosphorylates a deletion mutant of HEO which consists almost exclusively of the hormone binding domain. Using HEGO and HEO in parallel and measuring both binding activation and phosphorylation of ER we now observe that the wild type receptor is a good kinase substrate, slightly better than HEO. Furthermore, HEGO like the calf uterus receptor in the presence of estradiol, stimulates the kinase. From present findings it appears that ER and uterus tyrosine kinase are functionally associated and that this association is abolished by glycine to valine substitution at position 400 of ER.

In vitro interaction of estradiol receptor with Ca2+-calmodulin.

Estradiol receptor (ER) activity requires interaction with hormone and specific DNA sequence. We now report that this receptor also interacts with calmodulin (CaM), the major intracellular mediator of Ca2+ action in eucaryotic cells. This interaction has been observed using both CaM-Sepharose and [125I]CaM. Crude and purified [3H]ER complex show high affinity interaction with CaM-Sepharose [dissociation constant (Kd) 0.12 and 0.16 nM, respectively]. Unoccupied receptor shows a similar high affinity interaction. Tamoxifen-ER complex also binds to CaM-Sepharose. Several findings show that this CaM-ER interaction is very specific: lack of this interaction has been observed in the presence of trifluoperazine, an inhibitor of protein binding to CaM; the receptor binds neither Sepharose, nor parvalbumin-Sepharose; competition of interaction of [3H]ER complex with CaM-Sepharose is observed by cold ER complex; rat liver glucocorticoid receptor does not bind to CaM-Sepharose. The interaction of purified receptor with 125I-labeled CaM has been detected by various techniques: centrifugation through sucrose gradient of CaM incubated with receptor shows that CaM binds to a protein forming a complex sedimenting at 5 S. This complex is shifted to the 7.5 S region by a monoclonal antireceptor antibody. Incubation of CaM with receptor followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis fluorography of the immunoprecipitated receptor shows that [125I]CaM coprecipitates with the receptor. Competition of this interaction by an excess of cold CaM is observed. Interaction of the receptor with CaM is also observed by the overlay technique.(ABSTRACT TRUNCATED AT 250 WORDS)

Oestradiol stimulates tyrosine phosphorylation and hormone binding activity of its own receptor in a cell-free system.

Recent experiments have shown that calf uterus oestrogen receptor exists in a tyrosine-phosphorylated hormone binding form and in non-phosphorylated, non-hormone binding form. We report here that physiological concentrations of oestradiol in complex with the receptor stimulate the calf uterus receptor kinase that converts the non-hormone binding receptor into hormone binding receptor through phosphorylation of the receptor on tyrosine. The activity of this enzyme has been followed by reactivation of hormone binding sites and phosphorylation on tyrosine of calf uterus phosphatase-inactivated receptor. Phosphorylation of the receptor has been demonstrated by interaction of kinase 32P-phosphorylated proteins with anti-receptor antibody followed either by sucrose gradient centrifugation or SDS-PAGE of the immunoprecipitated proteins. Hormone stimulation of the kinase is inhibited by receptor occupancy of the anti-oestrogen tamoxifen. Oestradiol-receptor complex increases the affinity of the kinase for the dephosphorylated receptor. Findings of this report are consistent with the observation that several protein tyrosine kinases that are associated with peptide hormone receptors are stimulated by the binding of the hormone to the receptor. This is the first report on the activation of a tyrosine kinase by a steroid hormone. The finding that hormones can regulate their own receptor binding activity through a tyrosine kinase is also new.

Phosphorylation on tyrosine of oestradiol-17 beta receptor in uterus and interaction of oestradiol-17 beta and glucocorticoid receptors with antiphosphotyrosine antibodies.

In whole rat uterus incubated in the presence of [32P]orthophosphate the oestradiol receptor is [32P]phosphorylated on tyrosine. This finding follows our previous observation that in vitro this receptor can be phosphorylated on tyrosine by a uterus kinase that endows the receptor with oestradiol-binding activity. The calf uterus oestradiol receptor interacts with high affinity with 2G8 and 1G2 antiphosphotyrosine antibodies coupled to Sepharose (Kd values of 0.28 and 1.1 nM, respectively). The interaction with 2G8 antibody has been exploited to purify the oestradiol receptor. This interaction disappears after inactivation of the oestradiol receptor by the nuclear phosphatase that hydrolyses phosphotyrosine of the receptor. This fact substantiates the evidence that the oestradiol receptor in uterus is phosphorylated on tyrosine and that this phosphorylation is required for hormone binding to the receptor. The rat liver glucocorticoid receptor also interacts with high affinity with 2G8 antiphosphotyrosine antibody coupled to Sepharose (Kd value of 0.21 nM). This receptor has been purified by using in sequence heparin-Sepharose and antiphosphotyrosine antibody-Sepharose.

Estradiol and progesterone receptors in malignant gastrointestinal tumors.

Estradiol and progesterone receptors were assayed in tumors from 79 patients with primary colorectal and 56 patients with stomach adenocarcinomas. Eighteen of 79 colorectal cancers contained estradiol receptor, while 34 specimens were positive for progesterone receptor. In stomach cancer, the positive samples were 8 for estradiol and 14 for progesterone receptors. In both types of tumors, the Kd was in the range of 10(-10) M for estradiol and 10(-9) M for progesterone receptor, respectively. In colorectal adenocarcinomas, the presence of progesterone receptor seems to be partially correlated to the presence of estradiol receptor while, in stomach tumors, this correlation is lost. The positivity of at least one receptor in colorectal cancers is higher in the female sex. The contrary occurs for stomach cancer. Sucrose gradient centrifugation showed that cytoplasmic estradiol receptor of stomach cancer sedimented at 8S or 4 to 5S at low ionic strength. The isoelectric point of stomach cancer estradiol receptor is 6.5.

Estrogen and progesterone binding proteins in human colorectal cancer. A preliminary characterization of estradiol receptor.

Estradiol receptor (ER) and progesterone receptor (PgR) were assayed in tumors from 20 patients with primary colorectal cancer. Ten of 20 tumors contained high affinity sites for 17 beta-estradiol and progesterone. The highest concentration of ER was 56 fmol/mg of protein. The ER dissociation constant ranged from 1.6 X 10(-10) M (mean 4.6 +/- 2.6). The highest concentration of PgR was 42 fmol/mg of protein. The PgR dissociation constant ranged from 3 X 10(-9) to 9 X 10(-9) M (mean 5.65 +/- 2.1). Four out of 20 specimens analyzed were from male patients and all resulted negative for both receptors. Sixty per cent of ER positive tumors were also PgR positive, whereas only 20% of ER negative were PgR positive. Sucrose gradient centrifugation showed that cytoplasmic ER of colorectal cancer sedimented at 3 S in the absence of protease inhibitors and at 4.5 S in the presence of 1 mM phenylmethylsulphonyl fluoride (PMSF) both in low and in high ionic strength. When chromatographed on Sephadex G-200 almost all ER was quantitatively recovered in the included fractions. Molecular weights of ER eluted from Sephadex G-200 ranged from 90,000 to 50,000 daltons. Elution profile and molecular weight heterogeneity suggest that, in spite of the presence of PMSF, there is a limited proteolysis of ER. Partially purified colorectal cancer ER did not bind to sepharose-heparin. The isoelectric point of ER was 6.4-6.5.

Estrogen binding proteins of calf uterus. Molecular and functional characterization of the receptor transforming factor: A Ca2+-activated protease.

It was previously shown that calf uterus cytosol contains a Ca2+-activated receptor transforming factor (RTF) which irreversibly converts the larger molecular states of estrogen receptor (5.3 to 8.6 S, depending on ionic strength) into a smaller, salt-stable form (4.5 S, independent of ionic strength). We now describe a method for rapid and reliable separation of precursor and RTF-transformed receptor forms, which takes advantage of a difference in isoelectric point between the two: the more acidic precursor (isoelectric point, 6.2) is still retained by DEAE-cellulose under conditions (0.12 M KCl, pH 8.3) which produce release from cellulose of the less acidic transformed form (isoelectric point, 6.6 to 6.8). Based on this method of separation, RTF activity can be assayed easily and we could thus progress in the purification and physical and functional characterization of this factor, RTF has been purified about 100-fold. Molecular properties, as assayed by methods suited to partially purified preparations, are as follows: sedimentation coefficient, 6.4 S; Stokes radius, 45 A; molecular weight, 115,000; isoelectric point, 4.9. The Michaelis constant, expressed as moles/liter of estradiol binding sites, is 1.25 X 10(8), at pH 7.5 and 4 degrees, pH 8.5 is optimum for activity. RTF attacks native casein (Km, 1.25 X 10(-5) mol/liter at pH 7.5 and 22 degrees) but not hemoglobin, ovalbumin, or albumin. N-Benzoylarginine methyl ester is a competitive inhibitor of RTF-induced receptor transformation, while L-leucylglycylglycine and N-benzoyltyrosinamide are not. RTF activity is protected by -SH compounds. RTF activity is Ca2+-dependent. Ca2+ starts an activation-inactivation cycle of RTF, with permanent loss of transforming activity which proceeds at a particularly fast rate in the absence of substrate. Mg2+ is inactive, while Sr2+ and Mn2+ may in part substitute for Ca2+. RTF is present in both endometrium and myometrium. RTF is not a lysosomal hydrolase, as shown by its alkaline pH optimum (8.5) and exclusive location in cytosol, nor is it trypsin or a protease of the trypsin group. Also, it is distinct from known proteases of human uterus. The functional significance of this Ca2+-activated protease of cytosol with alkaline pH optimum and high affinity for the larger native form of receptor is still unknown.