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S Tozlu-Kara, V Roux, C Andrieu, J Vendrell, S Vacher, V Lazar, F Spyratos, M Tubiana-Hulin, P Cohen, P Dessen, R Lidereau and I Bièche

Abstract

The estrogen receptor α (ERα) status of breast tumors is used to identify patients who may respond to endocrine agents such as tamoxifen. However, ERα status alone is not perfectly predictive, and there is a pressing need for more reliable markers of endocrine responsiveness. In this aim, we used a two-step strategy. We first screened genes of interest by a pangenomic 44 K oligonucleotide microarray in a series of ten ERα-positive tumors from five tamoxifen-treated postmenopausal patients who relapsed (distant metastasis) and five tamoxifen-treated postmenopausal patients who did not relapse, matched with respect to age, Scarff–Bloom–Richardson grade, lymph node status, and macroscopic tumor size. Genes of interest (n=24) were then investigated in an independent well-characterized series of ERα-positive unilateral invasive primary breast tumors from postmenopausal women who received tamoxifen alone as adjuvant hormone therapy after primary surgery. We identified four genes (HRPAP20, TIMELESS, PTPLB, and MGC29814) for which high mRNA levels were significantly associated with shorter relapse-free survival (log-rank test). We also showed that hormone-regulated proliferation-associated 20 kDa protein (HRPAP20) and TIMELESS are 17β-estradiol-regulated in vitro and are ectopically expressed in OH-Tam-resistant cell lines. In conclusion, these findings point to HRPAP20 and TIMELESS as promising markers of tamoxifen resistance in women with ERα-positive breast tumors.

Free access

Nicola J Smith and Tim R Fenton

The interaction between human papillomaviruses (HPV) and the apolipoprotein-B mRNA-editing catalytic polypeptide-like (APOBEC)3 (A3) genes has garnered increasing attention in recent years, with considerable efforts focused on understanding their apparent roles in both viral editing and in HPV-driven carcinogenesis. Here, we review these developments and highlight several outstanding questions in the field. We consider whether editing of the virus and mutagenesis of the host are linked or whether both are essentially separate events, coincidentally mediated by a common or distinct A3 enzymes. We discuss the viral mechanisms and cellular signalling pathways implicated in A3 induction in virally infected cells and examine which of the A3 enzymes might play the major role in HPV-associated carcinogenesis and in the development of therapeutic resistance. We consider the parallels between A3 induction in HPV-infected cells and what might be causing aberrant A3 activity in HPV-independent cancers such as those arising in the bladder, lung and breast. Finally, we discuss the implications of ongoing A3 activity in tumours under treatment and the therapeutic opportunities that this may present.

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A. J. L. Clark, P. M. Lavender, G. M. Besser and L. H. Rees

ABSTRACT

As an approach to understanding the abnormalities of pro-opiomelanocortin (POMC) gene regulation in human ACTH-secreting tumours, we have analysed the POMC mRNA content of nine such tumours using the Northern blot technique. Most of the tumours and normal human pituitary contained easily detectable quantities of POMC mRNA. The length of this message in most tumours was similar to, or slightly larger than, that in the normal pituitary (1150–1200 bases). Ribonuclease H studies suggested that the origin of any size heterogeneity was a longer poly(A) tail in the tumour RNA. Some tumours, however, expressed a short POMC mRNA (800 bases) which may lack the first two exons of the POMC gene as has been described. A third POMC mRNA size variant (1500 bases) was also seen in low levels in two cases, and as the principal mRNA species in one case. Primer extension and S1 nuclease protection studies suggested that most transcripts in the tumours analysed originated from the conventional promoter, and thus the use of an alternative promoter is not an adequate explanation for the expression of this gene in ectopic ACTH-secreting tumours.

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Norman G Nicolson, Reju Korah and Tobias Carling

Adrenocortical carcinomas are rare tumors with poor prognosis and limited treatment options. Although widely used as in vitro models to test novel therapeutic strategies, the adrenocortical carcinoma-derived cell lines NCI-H295R and SW-13 have only partially been described genetically. Our aim was to characterize the mutational landscape of these cells to improve their experimental utility and map them to clinical subtypes of adrenocortical carcinoma. Genomic DNA from NCI-H295R and SW-13 cells was subjected to whole-exome sequencing. Variants were filtered for non-synonymous mutations and curated for validated adrenocortical and pan-cancer driver gene mutations. Genes mutated in the cell lines were mapped using gene ontology and protein pathway tools to determine signaling effects and compared to mutational and clinical characteristics of 92 adrenocortical carcinoma cases from The Cancer Genome Atlas. NCI-H295R and SW-13 cells carried 1325 and 1836 non-synonymous variants, respectively. Of these, 61 and 76 were known cancer driver genes, of which 32 were shared between cell lines. Variant interaction analyses demonstrated dominant TP53 dysregulation in both cell lines complemented by distinct WNT (NCI-H295R) and chromatin remodeling (SW-13) pathway perturbations. Both cell lines genetically resemble more aggressive adrenocortical carcinomas with worse prognosis, for which development of targeted therapies is most critical. Careful incorporation of the genetic landscapes outlined in this study will further the in vitro utility of these cell lines in testing for novel therapeutic approaches for adrenocortical malignancy.

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M Delhase, F Rajas, P Verdood, C Remy, P Chevallier, B Velkeniers, J Trouillas and E L Hooghe-Peters

ABSTRACT

We have combined different techniques to analyse passages of five different rat spontaneous pituitary tumours (SMtTW) that were transplanted under the kidney capsule. These tumours were secreting prolactin (PRL), GH or both hormones. RIA, immunocytochemistry (ICC) and Western blot analysis were applied to characterize the hormone(s) stored (ICC and Western blot) and secreted (RIA). mRNA content was analysed by PCR, Northern blot analysis and in situ hybridization.

The data point not only to the reliability of the techniques used at both protein and RNA levels for each tumour studied but also to the complementarity of some techniques. For example, whereas Northern blot analysis demonstrates the presence and size of hormone mRNA, in situ hybridization indicates the percentage of cells expressing a given hormone mRNA and allows the presence of one population (or more) of cells in a given tumour to be identified.

Moreover, the tumours were compared with normal rat pituitary. Although the PRL and GH mRNAs were identical in size, the amount of mRNA was lower in the tumours. At the protein level, the PRL and GH variants exhibited a different pattern of expression in tumours compared with the normal rat pituitary.

The biological significance of these differences is discussed.

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H M Fraser, S F Lunn, G M Cowen and P T K Saunders

ABSTRACT

Localization of inhibin/activin subunit mRNAs within the macaque ovary from the immediate pre-ovulatory period of the menstrual cycle, when serum immunoreactive inhibin begins to rise, to day 9 of the luteal phase, when serum inhibin concentrations are maximal, was investigated using in-situ hybridization. Ovaries were studied on the day of the LH surge (day 0) and on days 2, 5, and 9 of the luteal phase by hybridizing frozen tissue sections with radio-labelled riboprobes specific to the inhibin/activin α-, βA-and βB-subunits. After autoradiographic exposure for 10 and 21 days, grain concentrations were quantified by image analysis. Moderate expression of α-, βA- and βB-subunit mRNA was present within the granulosa cells of the pre-ovulatory follicle (day 0). The granulosa-lutein cells of the corpora lutea expressed high levels of α-subunit at days 2, 5 and 9. mRNAs for βA and βB were detected at low but significant levels in all of the corpora lutea. All healthy antral follicles exhibited a high level of expression of βB-subunit mRNA in the granulosa cells. On day 2 after ovulation these follicles also expressed high α- and moderate βA-subunit mRNA. On day 9 the βB-inhibin mRNA in antral follicles was found in association with low expression of the other subunits. Small follicles in ovaries on day 2 expressed moderate α- and low levels of βB-subunit mRNA, while mRNA for βA was absent. α-subunit mRNA expression was present on day 5 while neither βA- nor βB-subunit mRNA was detected. On day 9 a proportion of small follicles expressed α- and βA-subunit mRNA. These results demonstrate that marked differences are present in the levels of expression of the three inhibin/activin subunit genes between follicles and the corpus luteum. The predominance of the βB-subunit mRNA within antral follicles would be consistent with the synthesis of activin. The predominance of the α-subunit combined with the low expression of the β-subunits in the corpus luteum suggests that both biologically active inhibin and free α-subunit are produced by the primate corpus luteum.

Free access

E Karteris, N Papadopoulou, DK Grammatopoulos and EW Hillhouse

Corticotrophin-releasing hormone (CRH) has been identified in several peripheral tissues, including the female reproductive organs. CRH is expressed in the placenta, myometrium, epithelial endometrium and the endometrial stromal cells at all phases of the menstrual cycle. Similarly, CRH receptors are present in pregnant and non-pregnant myometrium, placenta and endometrium. Putative roles of CRH in the endometrium include involvement in implantation, decidualisation and maintenance of pregnancy. In this study we sought to investigate in detail the CRH receptor repertoire expressed in the human endometrium and their signalling characteristics. Using RT-PCR we were able to demonstrate the expression of CRH receptor 1alpha (CRH-R1alpha) and CRH-R2alpha in the human endometrium. CRH-R1beta was present in 40% of endometrial cDNAs examined. No apparent expression of CRH-R2beta, CRH-R2gamma or any other CRH-R1 splice variants was detected. Chemical cross-linking studies with 125I-ovine CRH revealed that the endometrial CRH receptor has a molecular weight of 45 kDa. Using the non-hydrolysable photoreactive analogue [alpha-32P]GTP-azidoanilide and peptide antisera raised against G-protein alpha-subunits, we then studied coupling of endometrial CRH receptors to G proteins. Treatment of endometrial membranes with human CRH (100 nM) increased the labelling of Gq and Gs, but not Gi or Go. These results were supported by experiments in epithelial cells of the non-pregnant human endometrium in the secretory phase which showed that CRH induced increases in both cAMP and inositol trisphosphate levels. These results suggested that CRH may exert multiple effects in the human endometrium via distinct signalling cascades. These events are possibly mediated via different receptor subtypes.

Free access

M Watanabe, ER Simpson, N Pathirage, S Nakajin and CD Clyne

A number of clinical studies have highlighted the importance of estrogen in bone growth and maintenance in men and postmenopausal women. In these instances, estrogen is synthesized locally within bone tissue by aromatase, encoded by the CYP19 gene. The mechanisms regulating aromatase expression in bone, however, are unclear. In this work we characterized the expression of aromatase activity and gene transcripts in the human fetal osteoblastic cell line, SV-HFO. Aromatase activity and gene transcript expression were stimulated by dexamethasone. Oncostatin M strongly stimulated aromatase expression in synergy with dexamethasone. These factors induced CYP19 transcripts that included the sequence of exon I.4 in their 5'UTR. Consistent with this, a reporter construct harboring the genomic sequence of the promoter region of exon I.4 (promoter I.4) was also activated by dexamethasone and oncostatin M. 5' deletion and mutation analysis revealed important roles for a glucocorticoid response element, an interferon gamma activating sequence and a putative binding site for Sp1. Transfection of exogenous glucocorticoid receptor, STAT3 or Sp1 increased promoter activity, indicating a potential role for these transcription factors in regulating aromatase expression in SV-HFO cells. These data suggest that the SV-HFO cell line is a valuable model with which to elucidate the mechanisms regulating local estrogen synthesis in osteoblasts.

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Carlos Larqué, Myrian Velasco, Francisco Barajas-Olmos, Neyvis García-Delgado, Juan Pablo Chávez-Maldonado, Jazmín García-Morales, Lorena Orozco and Marcia Hiriart

Research on the postnatal development of pancreatic beta-cells has become an important subject in recent years. Understanding the mechanisms that govern beta-cell postnatal maturation could bring new opportunities to therapeutic approaches for diabetes. The weaning period consists of a critical postnatal window for structural and physiologic maturation of rat beta-cells. To investigate transcriptome changes involved in the maturation of beta-cells neighboring this period, we performed microarray analysis in fluorescence-activated cell-sorted (FACS) beta-cell-enriched populations. Our results showed a variety of gene sets including those involved in the integration of metabolism, modulation of electrical activity, and regulation of the cell cycle that play important roles in the maturation process. These observations were validated using reverse hemolytic plaque assay, electrophysiological recordings, and flow cytometry analysis. Moreover, we suggest some unexplored pathways such as sphingolipid metabolism, insulin-vesicle trafficking, regulation of transcription/transduction by miRNA-30, trafficking proteins, and cell cycle proteins that could play important roles in the process mentioned above for further investigation.

Free access

JL Thomas, JI Mason, G Blanco and ML Veisaga

Human type I 3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD/isomerase) is an integral membrane protein of human placental trophoblast and of insect Sf9 cells transfected with recombinant baculovirus containing the cDNA encoding the enzyme. Purified native or wild-type enzyme remains in solution only in the presence of detergent that may prevent crystallization. The membrane-spanning domain (residues 283-310) of the enzyme protein was deleted in the cDNA using PCR-based mutagenesis. The modified enzyme was expressed by baculovirus in the cytosol instead of in the microsomes and mitochondria of the Sf9 cells. The cytosolic form of 3beta-HSD/isomerase was purified using affinity chromatography with Cibacron Blue 1000. The NAD(+) and NaCl used to elute the enzyme were removed by size-exclusion centrifugation. Hydroxylapatite chromatography yielded a 26-fold purification of the enzyme. SDS-PAGE revealed a single protein band for the purified cytosolic enzyme (monomeric molecular mass 38.8 kDa) that migrated just below the wild-type enzyme (monomeric molecular mass 42.0 kDa). Michaelis-Menten constants measured for 3beta-HSD substrate (dehydroepiandrosterone) utilization by the purified cytosolic enzyme (K(m)=4.5 microM, V(max)=53 nmol/min per mg) and the pure wild-type enzyme (K(m)=3.7 microM, V(max)=43 nmol/min per mg), for isomerase substrate (5-androstene-3,17-dione) conversion by the purified cytosolic (K(m)=25 microM, V(max)=576 nmol/min per mg) and wild-type (K(m)=28 microM, V(max)=598 nmol/min per mg) enzymes, and for NAD(+) reduction by the 3beta-HSD activities of the cytosolic (K(m)=35 microM, V(max)=51 nmol/min per mg) and wild-type (K(m)=34 microM, V(max)=46 nmol/min per mg) enzymes are nearly identical. The isomerase activity of the cytosolic enzyme requires allosteric activation by NADH (K(m)=4.6 microM, V(max)=538 nmol/min per mg) just like the wild-type enzyme (K(m)=4.6 microM, V(max)=536 nmol/min per mg). Crystals of the purified, cytosolic enzyme protein have been obtained. The inability to crystallize the detergent-solubilized, wild-type microsomal enzyme has been overcome by engineering a cytosolic form of this protein. Determining the tertiary structure of 3beta-HSD/isomerase will clarify the mechanistic roles of potentially critical amino acids (His(261), Tyr(253)) that have been identified in the primary structure.