Gilles Leblanc

A candidate prostate cancer susceptibility gene at chromosome 17p

It is difficult to identify genes that predispose to prostate cancer due to late age at diagnosis, presence of phenocopies within high-risk pedigrees and genetic complexity. A genome-wide scan of large, high-risk pedigrees from Utah has provided evidence for linkage to a locus on chromosome 17p. We carried out positional cloning and mutation screening within the refined interval, identifying a gene, ELAC2, harboring mutations (including a frameshift and a nonconservative missense change) that segregate with prostate cancer in two pedigrees. In addition, two common missense variants in the gene are associated with the occurrence of prostate cancer. ELAC2 is a member of an uncharacterized gene family predicted to encode a metal-dependent hydrolase domain that is conserved among eukaryotes, archaebacteria and eubacteria. The gene product bears amino acid sequence similarity to two better understood protein families, namely the PSO2 (SNM1) DNA interstrand crosslink repair proteins and the 73-kD subunit of mRNA 3′ end cleavage and polyadenylation specificity factor (CPSF73).

Evaluation of BRCA1 and BRCA2 mutation prevalence, risk prediction models and a multistep testing approach in French-Canadian families with high risk of breast and ovarian cancer

Background and objective: In clinical settings with fixed resources allocated to predictive genetic testing for high-risk cancer predisposition genes, optimal strategies for mutation screening programmes are critically important. These depend on the mutation spectrum found in the population under consideration and the frequency of mutations detected as a function of the personal and family history of cancer, which are both affected by the presence of founder mutations and demographic characteristics of the underlying population. The results of multistep genetic testing for mutations in BRCA1 or BRCA2 in a large series of families with breast cancer in the French-Canadian population of Quebec, Canada are reported. Methods: A total of 256 high-risk families were ascertained from regional familial cancer clinics throughout the province of Quebec. Initially, families were tested for a panel of specific mutations known to occur in this population. Families in which no mutation was identified were then comprehensively tested. Three algorithms to predict the presence of mutations were evaluated, including the prevalence tables provided by Myriad Genetics Laboratories, the Manchester Scoring System and a logistic regression approach based on the data from this study. Results: 8 of the 15 distinct mutations found in 62 BRCA1/BRCA2-positive families had never been previously reported in this population, whereas 82% carried 1 of the 4 mutations currently observed in ⩾2 families. In the subset of 191 families in which at least 1 affected individual was tested, 29% carried a mutation. Of these 27 BRCA1-positive and 29 BRCA2-positive families, 48 (86%) were found to harbour a mutation detected by the initial test. Among the remaining 143 inconclusive families, all 8 families found to have a mutation after complete sequencing had Manchester Scores ⩾18. The logistic regression and Manchester Scores provided equal predictive power, and both were significantly better than the Myriad Genetics Laboratories prevalence tables (p<0.001). A threshold of Manchester Score ⩾18 provided an overall sensitivity of 86% and a specificity of 82%, with a positive predictive value of 66% in this population. Conclusion: In this population, a testing strategy with an initial test using a panel of reported recurrent mutations, followed by full sequencing in families with Manchester Scores ⩾18, represents an efficient test in terms of overall cost and sensitivity.

Spironolactone-related inhibitors of type II 17β-hydroxysteroid dehydrogenase: chemical synthesis, receptor binding affinities, and proliferative/antiproliferative activities

The family of 17beta-hydroxysteroid dehydrogenases (17beta-HSDs) catalyzes the formation and inactivation of testosterone (T), dihydrotestosterone (DHT), and estradiol (E2), thus playing a crucial role in the regulation of active steroid hormones in target tissues. Among the five known 17beta-HSD enzymes, type II catalyzes the oxidation of E2 into estrone (E1), T into androstenedione, DHT into androstanedione, and 20alpha-dihydroprogesterone into progesterone. Specific inhibitors are thus an interesting means to study the regulation and to probe the structure of type II 17beta-HSD. In this context, we have efficiently synthesized a series of 7alpha-thioalkyl and 7alpha-thioaryl derivatives of spironolactone that inhibit type II 17beta-HSD. These new C19-steroidal inhibitors possess two important pharmacophores, namely 17-spiro-gamma-lactone and a bulky side-chain at the 7alpha-position. It was found that a para-substituted benzylthio group at the 7alpha-position enhances the inhibitory potency of spironolactone derivatives on type II 17beta-HSD. In fact, the compound with a para-hydroxy-benzylthio group showed an IC50 value of 0.5 microM against type II 17beta-HSD, whereas the compound with a para-[2-(1-piperidinyl)-ethoxy]-benzylthio group inhibited this enzyme with an IC50 value of 0.7 microM. The latter inhibitor is more selective than the former because it did not show any inhibitory potency against P450 aromatase as well as any affinity towards four steroid receptors (AR, PR, GR, ER). As a result, this inhibitor did not show any proliferative effect on androgen-sensitive Shionogi cells and estrogen-sensitive ZR-75-1 cells. These findings contribute to a better knowledge of the structure of type II 17beta-HSD and offer an interesting tool to study the regulation of this enzyme in several biological systems.

Binding Characteristics of Novel Nonsteroidal Antiestrogens to the Rat Uterine Estrogen Receptors

Tamoxifen (TAM), the only antiestrogen currently available for the endocrine therapy of breast cancer behaves as a mixed agonist/antagonist of estrogen action, thus limiting its therapeutic potential. We report the binding characteristics of a novel series of nonsteroidal antiestrogens to the rat uterine estrogen receptor. As measured by competition studies, the affinity of EM-652, the active metabolite of the prodrug EM-800, for the estrogen receptor is 7-11 times higher than that of 17beta-estradiol (E2), ICI 182780, and hydroxy-tamoxifen (OH-TAM), the active metabolite of Tamoxifen. EM-652 is 20x more potent than ICI 164384 and Droloxifene while it is 400 times more potent than Toremifene in displacing [3H]E2 from the rat uterine estrogen receptor. On the other hand, the prodrug EM-800 and Tamoxifen have respectively 150-fold and 410-fold less affinity for the estrogen receptor than the pure antiestrogen EM-652. No significant binding of EM-652, EM-800, TAM or OH-TAM was observed to the rat uterine progesterone receptor at concentrations up to 10,000 nM except for TAM that caused a 50% displacement of labeled R5020 at 4000 nM. No significant binding of EM-652 or EM-800 was observed on the rat ventral prostate androgen receptor or the rat uterine progesterone receptor. The present data demonstrate the high affinity and specificity of the new antiestrogen, EM-652, for the rat uterine estrogen receptor. The antiestrogen EM-652 thus becomes the compound having the highest known affinity for the estrogen receptor. Due to its unique potency and its pure antiestrogenic activity already demonstrated in many systems, this antiestrogen could well offer an important advance for the endocrine therapy of breast cancer, uterine cancer, and other estrogen-sensitive diseases in women.

Purification, Cloning, Complementary DNA Structure, and Predicted Amino Acid Sequence of Human Estradiol 17β‐Dehydrogenase

Seventeen-P-hydroxysteroid dehydrogenase ( 17P-HSD) is the enzyme that catalyzes the interconversion of dehydroepiandrosterone (DHEA) and Sandrostenediol, 4-androstenedione and testosterone, and estrone and estradiol. This enzyme is present in many tissues such as the placenta,’,? testis,3 kidney,4 liver,’ skin? and ovary.’ This enzyme is likely to play a key role in the regulation of intracellular concentrations of sex hormones. The androgen testosterone and the estrogen 17Pestradiol are known to play an essential role in the development, growth, and function of all tissues responsible for reproduction and fertility in men and women. Moreover, sex steroids are involved in the regulation of hormone-sensitive cancer growth, especially prostate,* b r e a ~ t , ~ and endometrial cancer.IO Despite its purification from many tissues”-I4 and its partial biochemical chara c t e r i ~ a t i o n , ~ ~ ’ ~ little was known about the structure of this enzyme. We report the purification, cloning, and deduced amino acid sequence of estradiol 17pdehydrogenase (E2DH) from human placenta. The availability of cDNAs for EzDH offers the opportunity of studying in detail the factors controlling the expression of this crucial enzyme not only in gonadal but also in several peripheral estrogen target tissues.

Characterization of antisera to a partially purified prolactin receptor: effect on prolactin binding in different target tissues

Prolactin (PRL) receptors have been purified by affinity chromatography using a lactogenic hormone (human growth hormone, hGH) coupled to Affigel-10. The mean binding capacity of 3 separate purifications was 1.18 +/- 0.26 nmoles prolactin per mg protein, representing a 836-fold purification over crude microsomes and 4000-5000-fold over mammary gland homogenates and 16% purity. The receptor was characterized by HPLC using a TSK-SW-4000 and 3000 column connected in series and eluted in 0.1 M borate buffer containing 0.2 M NaCl and 0.1% Triton. A single peak of [125I]hGH binding activity with a retention time of 45.2 min (22.6 ml), representing an apparent molecular weight of 133000, was observed. A single peak of activity was also observed following polyacrylamide gel electrophoresis of the purified receptor in 0.1% Triton coincident with the Coomassie-stained protein band. Antibodies to the partially purified receptor preparations were prepared in sheep, goats and guinea pigs. Antisera to the prolactin receptor prepared in all three species were capable of inhibiting the binding of 125I-labeled ovine prolactin to receptors from rabbit mammary glands. Significant inhibition of binding was observed at antisera dilution of 1:10 000 with the sheep antiserum being the most potent (half-maximal inhibition (IM50) = 1:5700). All three antisera were able to inhibit PRL binding completely, but failed to affect labeled insulin or hCG binding and had very little effect on bGH binding. The specificity of the sheep antiserum was tested in rabbit mammary glands, ovary, adrenal, pig mammary glands and 8 rat tissues which contain PRL receptors. The antiserum was able to inhibit the binding of labeled PRL in all tissues, with the inhibition curves for the rat tissues being non-parallel when compared to rabbit mammary gland, suggesting a homology but not a complete identity between PRl receptors in various tissues and animal species. These studies demonstrate that prolactin receptors can be purified from rabbit mammary tissue and that antisera can be produced in several species. In addition, the binding studies suggest that in the various tissues the receptor molecule is more or less exposed to interaction with the antisera, or that the receptor protein differs somewhat between species.

Mutation analysis and characterization of HSD17B2 sequence variants in breast cancer cases from French Canadian families with high risk of breast and ovarian cancer

Estrogen exposure is a risk factor for breast cancer. Given that HSD17B2 gene encodes an enzyme that catalyses estradiol inactivation, it appears as a good candidate breast cancer susceptibility gene. This study was designed to screen for HSD17B2 germline mutations potentially involved in breast cancer predisposition. Our re-sequencing analysis did not identify any deleterious germline mutations, and therefore mutations in HSD17B2 do not explain the clustering of breast cancer cases in non-BRCA1/2 high-risk French Canadian families. However, six sequence variants were identified, including two novel missense variants. Expression assays revealed that p.Ala111Asp and p.Gly160Arg did not alter the catalytic properties of 17beta-hydroxysteroid dehydrogenase type 2 enzyme, although p.Ala111Asp appears to affect protein stability resulting in significant decreases in the protein levels, providing valuable information on structure-function relationship.

Preferential Establishment of a Slowly Dissociable Component in Plasma Membrane Compared to Intracellular Prolactin Receptors

Abstract Prolactin dissociates more readily from rat liver than from rabbit mammary prolactin receptors. The rate of dissociation is dependent in the time of association. In rat liver, prolactin dissociates from receptors at the cell periphery (plasma membrane, PM) more rapidly than those of microsomes, a major component of which is the intracellular Golgi membranes. The dissociation curves following 1 hr of association can be resolved into a fast and slow component by logarithmic transformation, with a greater than twofold increase in the fast componment of the dissociation rate constant (k 2) in PM compared to microsomal membranes. With longer association times (10 hr), plasma membranes develop a more slowly dissociable component, with dissociation characteristics (rate constants) similar to those observed in microsomes following 1 hr of association. Another means of visualizing this difference is by a Scatchard plot of prolactin binding to PM, microsomal, and Golgi membranes. The affinity constants in the microsomal and Golgi fractions are identical whereas that for the PM fractions more than twofold lower. The decreased affinity in the PM would allow prolactin to more readily dissociate from its receptor than from receptors with higher affinity. Although the differences between PM and Golgi receptors observed are small, they are confirmed by direct measurements of affinity constants and dissociation rate constants. Therefore, it appears that receptors with lower affinity at the cell periphery are those involved in the initial mechanism of action of prolactin.

Erratum: Evaluation of BRCA1 and BRCA2 mutation prevalence, risk prediction models and a multistep testing approach in French-Canadian families with high risk of breast and ovarian cancer (Journal of Medical Genetics (2007) 44, (107-121))

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Isolation and Sequence of the Human Glucocorticoid Receptor Gene Promoter. Cloning of the Human Androgen Receptor cDNA

We have isolated the promoter region of the human glucocorticoid receptor (hGR) gene from the λEMBL-3 human genomic library using synthetic oligonucleotides corresponding to the 5’ end of hGR cDNA. The clone was fine mapped by restriction digestion, hybridization with hGR cDNA probes and by DNA sequence analysis and found to contain an open reading frame corresponding to the hGR cDNA from amino acids 1–131 separated by a large intron from the 5’ noncoding sequences. The promoter region of the hGR has been identified by primer extension, nuclease S1 mapping using hGR mRNA from human prostatic carcinoma cells (LNCaP) and human breast tumor cells (MCF-7), by in vitro transcription using hGR-DNA fragments as template and by DNA sequence analysis. The sequence of the hGR promoter reveals that it contains neither a “TATA box” nor a “CART box” and has an extremely high “G+C” content. Gene transfer studies with hGR-CAT chimeric plasmids into COS-cells showed that the hormonal regulatory sequences of hGR is contained within a 4 kb EcoRl-XbaI fragment.