Cell density is a critical determinant of aromatase expression in adipose stromal cells
Obesity is associated with an increased risk of breast cancer among postmenopausal women. This is at least partly due to excessive estrogen production in adipose tissue of obese women. Aromatase, the key enzyme in estrogen biosynthesis, is an important target in endocrine therapy for estrogen receptor (ER)-positive postmenopausal breast cancer. In this study we show that high confluency of human adipose stromal cells (ASCs) cultured in vitro can significantly stimulate aromatase gene expression and reduce the expression of breast tumor suppressor BRCA1 and members of the NR4A orphan nuclear family. Furthermore, small interfering RNA (siRNA)-mediated knockdown of Nurr1, a member of the NR4A family, substantially increased aromatase expression. Lastly, we found that the cell density-triggered inducibility of aromatase expression varies in ASCs isolated from different disease-free individuals. Our finding highlights the impact of increased cell number on estrogen biosynthesis as in the case of excessive adiposity.

title: Mechanical phenotype is important for stromal aromatase expression
abstract: Evidence that aromatase expression in tumor-associated breast stroma is elevated, provides a rationale for use of aromatase inhibitors (AIs) in breast cancer treatment. However, regulation of local aromatase expression in cancer-free breast stroma is poorly understood. Recent clinical work indicates that stromal cells in dense breast tissue tend to express higher levels of aromatase than their counterpart from non-dense tissue. Consistent with the clinical observation, our cell culture-based study indicated that cell density, cell shape, and extracellular matrix (ECM) significantly induced stromal aromatase expression via a distinct signal transduction pathway. In addition, we identified a number of cell surface markers that are commonly associated with aromatase-expressing stromal cells. As mammographic density is one of the strongest and most prevalent risk factors for breast cancer, these findings provide a potential mechanistic link between alterations in tissue composition of dense breast tissue and increased stromal aromatase expression. Further exploration of the in vitro model system may advance understanding of an important problem in breast cancer biology.

title: Social deprivation and primary hyperparathyroidism.
abstract: To investigate the potential relationship between social status or deprivation and the prevalence of primary hyperparathyroidism (PHPT).



We retrospectively identified a cohort of patients diagnosed as having PHPT between 1981 and 2007 from the Scottish Morbidity Records database. The Scottish Index of Multiple Deprivation (SIMD) 2006 quintiles were derived for these patients by using the postal codes. The distribution of the SIMD quintiles was examined to determine the possible influence of deprivation on the incidence of PHPT.



In Scotland between 1981 and 2007, 3,039 patients were diagnosed as having PHPT, in accordance with the International Classification of Diseases code for PHPT. The distribution of the PHPT cohort across the SIMD 2006 quintiles was significantly different from that expected, with a higher representation (27.2%) among the most deprived and a lower representation (14.5%) in the least deprived quintile, in comparison with the 20% expected in each quintile (P<.0001).



The findings in this study suggest that socioeconomic deprivation is associated with an increased risk of developing PHPT.

title: Mutational Analysis of the Tetrahydrobiopterin-binding Site in Inducible Nitric-oxide Synthase*
abstract: Inducible nitric-oxide synthase (iNOS) is a hemeprotein that requires tetrahydrobiopterin (H4B) for activity. The influence of H4B on iNOS structure-function is complex, and its exact role in nitric oxide (NO) synthesis is unknown. Crystal structures of the mouse iNOS oxygenase domain (iNOSox) revealed a unique H4B-binding site with a high degree of aromatic character located in the dimer interface and near the heme. Four conserved residues (Arg-375, Trp-455, Trp-457, and Phe-470) engage in hydrogen bonding or aromatic stacking interactions with the H4B ring. We utilized point mutagenesis to investigate how each residue modulates H4B function. All mutants contained heme ligated to Cys-194 indicating no deleterious effect on general protein structure. Ala mutants were monomers except for W457A and did not form a homodimer with excess H4B and Arg. However, they did form heterodimers when paired with a full-length iNOS subunit, and these were either fully or partially active regarding NO synthesis, indicating that preserving residue identities or aromatic character is not essential for H4B binding or activity. Aromatic substitution at Trp-455 or Trp-457 generated monomers that could dimerize with H4B and Arg. These mutants bound Arg and H4B with near normal affinity, but Arg could not displace heme-bound imidazole, and they had NO synthesis activities lower than wild-type in both homodimeric and heterodimeric settings. Aromatic substitution at Phe-470 had no significant effects. Together, our work shows how hydrogen bonding and aromatic stacking interactions of Arg-375, Trp-457, Trp-455, and Phe-470 influence iNOSox dimeric structure, heme environment, and NO synthesis and thus help modulate the multiple effects of H4B.

title: Long term results of surgery versus continuous hyperfractionated accelerated radiotherapy (CHART) in patients aged >70 years with stage 1 non-small cell lung cancer.
abstract: Patients with T1N0 non-small cell lung cancer (NSCLC) are preferably treated by anatomic lobectomy. However, not all such patients are suitable for lobectomy due to their age or co-morbidity. Our aim was to determine the results obtained following lobectomy, wedge resection (WR) or continuous hyperfractionated accelerated radiotherapy (CHART) in patients aged >70 years.



Two hundred and fifteen consecutive patients aged >70 years, with pathologic stage 1 NSCLC in our unit between 1991 and 2001 were studied. Of these patients, 149 had a lobectomy, 47 had a WR and 19 had CHART. Follow-up was 100% complete.



Analysis demonstrated the WR and CHART patients to have reduced pulmonary function (FEV(1) 59% and 52%, respectively, of predicted vs. 76%, P<0.001) when compared to the lobectomy group but there were no differences among the groups with regard to mean age and histologic tumour type. There were no operative mortality among patients after WR; however, a 2.7% 30-day operative mortality among patients undergoing lobectomy (P=0.29). Kaplan-Meier survival curves at 1 and 5 years for patients undergoing WR, lobectomy and CHART was 98% and 74% vs. 97% and 68% vs. 80% and 39%, respectively (P=0.0484). The frequency of local/regional recurrence in the WR group (19.1%) was not significantly higher than in the lobectomy group (18.4%, P=0.38) when compared to the CHART group (27%, P=0.07).



Loco-regional recurrence and survival after WR and lobectomy in elderly patients with stage I NSCLC are comparable. Although the numbers are small, these data suggest that CHART is a reasonable treatment option for those who are not suitable candidates for surgery.

title: Cholesteryl ester hydrolase in human monocyte/macrophage: cloning, sequencing, and expression of full-length cDNA.
abstract: The sensitive technique of RT-PCR was used to identify cholesteryl ester hydrolase (CEH) expressed in human macrophages. This enzyme is thought to regulate the availability of intracellular free cholesterol for efflux. The expected 667-bp product was obtained starting with RNA from human peripheral blood and THP-1 monocytes and macrophages. The cDNA for human macrophage CEH was then cloned by PCR-based screening of a lambda-gt11 cDNA library. The full-length cDNA was sequenced and found to exhibit 76% homology (at the nucleotide and conceptually translated protein level) to hepatic CEH, an enzyme shown to be involved in hepatic cholesterol homeostasis and regulated by cholesterol at the transcription level via sterol response elements in the proximal promoter. Identification of the conserved catalytic triad (Ser(221, His(468), and Glu354)) and the SEDCLY motif places human macrophage CEH in the family of carboxylesterases. A greater than 20-fold increase in CEH activity was observed when COS-1 and COS-7 cells were transiently transfected with an eukaryotic expression vector, pcDNA3.1/V5/His-TOPO, containing the cDNA for human macrophage CEH. Using this full-length cDNA as a probe, a 2.2-kb transcript was identified by Northern blot analysis of total RNA from human peripheral blood and THP -1 macrophages. Overexpression of human macrophage CEH resulted in an impairment of upregulation of low-density lipoprotein (LDL) receptor mRNA in Chinese hamster ovary (CHO-K1) cells grown in cholesterol-deficient environment. These data identify the human macrophage CEH, demonstrate its expression in human peripheral blood macrophage and human macrophage cell line, THP-1, and suggest its role in the intracellular cholesteryl ester metabolism.

title: Early steps in reverse cholesterol transport: cholesteryl ester hydrolase and other hydrolases.
abstract: Several controversies exist related to the molecular identity and subcellular localization of the enzyme catalyzing macrophage cholesteryl ester hydrolysis. Some of these issues have been reviewed earlier and this review summarizes new developments that describe effects of overexpression or gene ablation. The main objective is to highlight the disagreement between lack of gene expression and incomplete abolition of macrophage cholesteryl ester hydrolytic activity and to emphasize the importance of redundancy.



New information resulting from the continuing characterization of the various cholesteryl ester hydrolases (hormone-sensitive lipase, HSL; cholesteryl ester hydrolase, CEH; and KIAA1363/NCEH1) is reviewed. Whereas CEH overexpression leads to beneficial effects such as decreased inflammation, improved glucose tolerance/insulin sensitivity, and attenuation of atherosclerotic lesion progression, deficiency/ablation of HSL or KIAA1363/NCEH1 results in incomplete loss of macrophage cholesteryl ester hydrolysis/turnover. New paradigms challenging the classical view of cytoplasmic cholesteryl ester hydrolysis and reverse cholesterol transport are also presented.



The observed beneficial effects of CEH overexpression identify macrophage cholesteryl ester hydrolysis as an important therapeutic target and future studies will determine whether similar effects are obtained with overexpression of HSL or KIAA1363/NCEH1. It is imperative that, for clinical benefit, mechanisms to enhance endogenous cholesteryl ester hydrolase(s) are established.