Christoforos Giatzakis - Academia.edu (original) (raw)

Papers by Christoforos Giatzakis

Nature Genetics, Jun 11, 2006

Supplementary Table 1 from Adrenal Hyperplasia and Adenomas Are Associated with Inhibition of Pho... more Supplementary Table 1 from Adrenal Hyperplasia and Adenomas Are Associated with Inhibition of Phosphodiesterase 11A in Carriers of PDE11A Sequence Variants That Are Frequent in the Population

Several types of adrenocortical tumors that lead to Cushing syndrome may be caused by aberrant cy... more Several types of adrenocortical tumors that lead to Cushing syndrome may be caused by aberrant cyclic AMP (cAMP) signaling. We recently identified patients with micronodular adrenocortical hyperplasia who were carriers of inactivating mutations in the 2q-located phosphodiesterase 11A (PDE11A) gene. We now studied the frequency of two missense substitutions, R804H and R867G, in conserved regions of the enzyme in several sets of normal controls, including 745 individuals enrolled in a longitudinal cohort study, the New York Cancer Project. In the latter, we also screened for the presence of the previously identified PDE11A nonsense mutations. R804H and R867G were frequent among patients with adrenocortical tumors; although statistical significance was not reached, these variants affected significantly enzymatic function in vitro with variable increases in cAMP and/or cyclic guanosine 3′,5′-monophosphate levels in HeLa and HEK293 cells. Adrenocortical tissues carrying the R804H mutatio...

Supplementary Figure 1 from Adrenal Hyperplasia and Adenomas Are Associated with Inhibition of Ph... more Supplementary Figure 1 from Adrenal Hyperplasia and Adenomas Are Associated with Inhibition of Phosphodiesterase 11A in Carriers of PDE11A Sequence Variants That Are Frequent in the Population

Carney complex (CC), Peutz-Jeghers syndrome (PJS), Cowden disease (CD), and Bannayan-Zonana syndr... more Carney complex (CC), Peutz-Jeghers syndrome (PJS), Cowden disease (CD), and Bannayan-Zonana syndrome (BZS) share clinical features, such as mucocutaneous lentigines and multiple tumors (thyroid, breast, ovarian, and testicular neoplasms), and autosomal dominant inheritance. A genetic locus has been identified for CC on chromosome 2 (2p16), and the genes for PJS, CD, and BZS were recently identified; genetic heterogeneity appears likely in both CC and PJS. The genes for PJS and CD/BZS, STK11/LKB1 and PTEN, respectively, may act as tumor suppressors, because loss of heterozygosity

Biochemistry, 2003

Peripheral-type benzodiazepine receptor (PBR) is an 18 kDa high-affinity drug ligand and choleste... more Peripheral-type benzodiazepine receptor (PBR) is an 18 kDa high-affinity drug ligand and cholesterol binding protein involved in various cell functions. Antisera for distinct PBR areas identified immunoreactive proteins of 18, 40, and 56 kDa and occasionally 72, 90, and 110 kDa in testicular Leydig and breast cancer cells. These sizes may correspond to PBR polymers and correlated to the levels of reactive oxygen species. Treatment of Leydig cells with human chorionic gonadotropin rapidly induced free radical, PBR polymer, and steroid formation. UV photoirradiation generates ROS species, which increased the size of intramembraneous particles of recombinant PBR reconstituted into proteoliposomes consistent with polymer formation, determined both by SDS-PAGE and by freeze-fracture electron microscopy. Spectroscopic analysis revealed the formation of dityrosines as the covalent cross-linker between PBR monomers. Moreover, photoirradiation increased PK 11195 drug ligand binding and reduced cholesterol binding capacity of proteoliposomes. Further addition of PK 11195 drug ligand to polymers increased the rate of cholesterol binding. These data indicate that reactive oxygen species induce in vivo and in vitro the formation of covalent PBR polymers. We propose that the PBR polymer might be the functional unit responsible for ligand-activated cholesterol binding and that PBR polymerization is a dynamic process modulating the function of this receptor in cholesterol transport and other cell-specific PBR-mediated functions.

Biochemistry, 2007

The translocator protein (18kDa; TSPO), previously known as peripheral-type benzodiazepine recept... more The translocator protein (18kDa; TSPO), previously known as peripheral-type benzodiazepine receptor, is a high affinity cholesterol-and drug-binding mitochondrial protein involved in various cell functions including steroidogenesis, apoptosis, and proliferation. TSPO is highly expressed in secretory and glandular tissues, especially in steroidogenic cells, and its expression is altered in certain pathological conditions such as cancer and neurological diseases. In this study, we characterized the regulatory elements present in the region of the TPSO promoter extending from 515 to 805-bp upstream of the transcription start site, an area previously identified as being important for transcription. Promoter fragments extending 2.7-kb and 805-bp upstream of the transcription start site were able to direct enhanced green fluorescent protein expression to Leydig cells of the testis, theca cells of the ovary, and cells of the adrenal cortex in transgenic animals. This expression pattern perfectly mimicked endogenous TSPO expression. Functional characterization of the 515 to 805-bp region revealed the presence of one Specificity protein 1/Specificity protein 3 (Sp1/Sp3) and two vets erythroblastosis virus E26 oncogene homolog (Ets) binding sites that are important for transcriptional activity in both MA-10 mouse Leydig tumor cells and NIH/3T3 whole mouse embryo fibroblasts. GA-binding protein α (GABPα)-a member of the Ets family of transcription factorswas found to be associated with the endogenous TSPO promoter. We conclude that Sp1/Sp3 and members of the Ets family of transcription factors bind to specific binding sites in the TSPO promoter to drive basal TSPO gene transcription.

Supplementary Figure Legend from Adrenal Hyperplasia and Adenomas Are Associated with Inhibition ... more Supplementary Figure Legend from Adrenal Hyperplasia and Adenomas Are Associated with Inhibition of Phosphodiesterase 11A in Carriers of PDE11A Sequence Variants That Are Frequent in the Population

Biochemistry, 2008

Translocator protein (TSPO) is an 18-kDa cholesterol-binding protein that is expressed at high le... more Translocator protein (TSPO) is an 18-kDa cholesterol-binding protein that is expressed at high levels in steroid synthesizing and several cancer cells where it is involved in steroidogenesis and cell proliferation, respectively. The factors regulating Tspo expression are unknown. We analyzed Tspo transcriptional responses to the tumor promoter, phorbol-12-myristate 13-acetate (PMA), in cells with varying TSPO levels. PMA induced Tspo promoter activity and Tspo mRNA levels in TSPO-poor non-steroidogenic cells (NIH-3T3 fibroblasts and COS-7 kidney), but not in TSPO-rich steroidogenic cells (MA-10 Leydig) with high basal Tspo transcriptional activity. The stimulatory effect of PMA was mediated by an 805-515-bp region upstream of the transcription start site. Electrophoretic mobility shift assay (EMSA) revealed that PMA induced binding of c-jun and GAbinding protein transcription factor (GABP-α) to their respective activator protein 1(AP1) and vets erythroblastosis virus E26 oncogene homolog (Ets) sites in this region. Protein kinase C (PKC)specific inhibitors blocked PMA induction of Tspo promoter activity with an inhibition profile suggestive of involvement of PKCε. PKCε expression correlated with TSPO content in the three cell lines. In NIH-3T3 cells, PKCε overexpression induced Tspo promoter activity, mRNA levels and enhanced PMA-induced up regulation of c-jun and TSPO. In MA-10 cells, a PKCε-specific translocation inhibitor peptide reduced basal Tspo promoter activity. PKCε siRNA pool reduced PKCε and TSPO levels in MA-10 cells indicating a role for PKCε in regulating TSPO expression. Taken together, these data suggest that elevated TSPO expression in steroidogenic cells maybe due to high constitutive expression of PKCε that renders them unresponsive to further induction while PMA activation of PKCε drives inducible TSPO expression in non-steroidogenic cells, likely through AP1 and Ets. The Translocator Protein (18kDa;TSPO), formerly known as the Peripheral-Type Benzodiazepine Receptor, is a high-affinity drug-and cholesterol-binding protein that was first identified in 1977 as an alternative binding site in the kidney for the benzodiazepine diazepam (1,2). TSPO binds various classes of organic compounds, including isoquinoline carboxamides such as PK11195 (3). TSPO is found in most tissues, although its expression among each tissue varies considerably (1,3,4). Secretory and glandular tissues, especially steroid hormone producing cells, are particularly rich in TSPO (1). Intermediate levels of this protein are found † This work was supported by Grant R01 ES07747 from the National Institutes of Health (to V.P.). V.P. was also supported by a Canada Research Chair in Biochemical Pharmacology. The Research Institute of MUHC is supported in part by a Center grant from Le Fonds de la recherche en santé du Québec.

ALTHOUGH several genes (reviewed in Ref. 1) have been investigated in adrenal tumorigenesis, the ... more ALTHOUGH several genes (reviewed in Ref. 1) have been investigated in adrenal tumorigenesis, the genetic background of adrenocortical tumors (ACT) remains poorly characterized. Adrenocortical hyperplasia is a polyclonal process, but ACT are mostly monoclonal lesions (2), indicating that genetic changes at specific loci in the genome are needed for adrenal tumorigenesis. A number of chromosomal abnormalities have been implicated in this process, including genomic loci on 11p and 17p (3–6), which harbor genes with tumor suppression or oncogenic function in the adrenal cortex. These include the genes coding for the p53 (on

Thyroid, 2010

Activating germline mutations of the RET gene cause multiple endocrine neoplasia type 2 and famil... more Activating germline mutations of the RET gene cause multiple endocrine neoplasia type 2 and familial medullary thyroid carcinoma (FMTC), conditions that are inherited in an autosomal dominant manner. In addition, somatic RET mutations have been identified in a variable proportion (about 30-70%) of sporadic (nonfamilial) MTC cases. We describe a Greek family with two novel likely pathogenic sequence variants of the RET gene. The first is a C to T transition at position 2458 (c.2458C&amp;amp;amp;amp;amp;amp;amp;amp;amp;gt;T) that causes an arginine to cysteine substitution (p.R820C) in exon 14 in the intracellular region of the kinase. This sequence variant was identified in an apparently healthy woman who had a recently deceased sister with confirmed aggressive MTC (age of onset 37 years). To assess the pathogenicity of this novel missense sequence variant, screening was performed on all available relatives: her two sons, the mother, and a second sister, including an MTC tumor sample from the deceased sister of the proband. At the time of the investigation, no clinical symptoms suggestive of multiple endocrine neoplasia type 2 or MTC were present in any of the individuals screened. The c.2458C&amp;amp;amp;amp;amp;amp;amp;amp;amp;gt;T transition was found in one son, the living sister, and the mother. Interestingly, it was not present in the tumor sample from the deceased sister. Instead, an in-frame deletion of 54 nt in exon 10 resulting in a protein missing 18 amino acids from I590 to G608 (c.1766_1819del 54) was found. Both genetic alterations were present in heterozygous state. These data suggest that the novel in-frame deletion was the disease-causing mutation in the deceased sister. The effect of the 2458C&amp;amp;amp;amp;amp;amp;amp;amp;amp;gt;T mutation on the activity of the kinase is under investigation.

Cancer Genetics and Cytogenetics, 1997

Hormone and Metabolic Research, 2008

Bloom syndrome is a genetic disorder associated with chromosomal instability and a predisposition... more Bloom syndrome is a genetic disorder associated with chromosomal instability and a predisposition to tumors that is caused by germline mutations of the BLM gene, a RecQ helicase. Benign adrenocortical tumors display a degree of chromosomal instability that is more significant than benign tumors of other tissues. Cortisol-producing hyperplasias, such as primary pigmented nodular adrenocortical disease (PPNAD), which has been associated with protein kinase A (PKA) abnormalities and/or PRKAR1A mutations, also show genomic instability. Another RecQ helicase, WRN, directly interacts with the PRKAR1B subunit of PKA. In this study, we have investigated the PRKAR1A expression in primary human Bloom syndrome cell lines with known BLM mutations and examined the BLM gene expression in PPNAD and other adrenal tumor tissues. PRKAR1A and other protein kinase A (PKA) subunits were expressed in Bloom syndrome cells and their level of expression differed by subunit and cell type. Overall, fibroblasts exhibited a significant decrease in protein expression of all PKA subunits except for PRKAR1A, a pattern that has been associated with neoplastic transformation in several cell types. The BLM protein was upregulated in PPNAD and other hyperplasias, compared to samples from normal adrenals and normal cortex, as well as samples from cortisol- and aldosterone-producing adenomas (in which BLM was largely absent). These data reveal an inverse relationship between BLM and PRKAR1A: BLM deficiency is associated with a relative excess of PRKAR1A in fibroblasts compared to other PKA subunits; and PRKAR1A deficiency is associated with increased BLM protein in adrenal hyperplasias.

Although several genes have been investigated in adrenal tumor- igenesis, the genetic background ... more Although several genes have been investigated in adrenal tumor- igenesis, the genetic background of adrenocortical tumors (ACT) re- mains poorly characterized. In southern Brazil, the annual incidence of ACT is unusually high, ranging from 3.4 - 4.2/million children, compared with a worldwide incidence of 0.3/million children younger than 15 yr. Environmental factors have been implicated because the distribution of these

Nature Genetics, 2000

Carney complex (CNC) is a multiple neoplasia syndrome characterized by spotty skin pigmentation, ... more Carney complex (CNC) is a multiple neoplasia syndrome characterized by spotty skin pigmentation, cardiac and other myxomas, endocrine tumours and psammomatous melanotic schwannomas 1-5 . CNC is inherited as an autosomal dominant trait and the genes responsible have been mapped to 2p16 and 17q22-24 (refs 6,7). Because of its similarities to the McCune-Albright syndrome 5,8 and other features, such as paradoxical responses to endocrine signals 9 , genes implicated in cyclic nucleotide-dependent signalling have been considered candidates for causing CNC (ref. 10). In CNC families mapping to 17q, we detected loss of heterozygosity (LOH) in the vicinity of the gene (PRKAR1A) encoding protein kinase A regulatory subunit 1-α (RIα), including a polymorphic site within its 5´ region. We subsequently identified three unrelated kindreds with an identical mutation in the coding region of PRKAR1A. Analysis of additional cases revealed the same mutation in a sporadic case of CNC, and different mutations in three other families, including one with isolated inherited cardiac myxomas. Analysis of PKA activity in CNC tumours demonstrated a decreased basal activity, but an increase in cAMP-stimulated activity compared with non-CNC tumours. We conclude that germline mutations in PRKAR1A, an apparent tumour-suppressor gene, are responsible for the CNC phenotype in a subset of patients with this disease.

Case Reports, 2009

Von Hippel-Lindau disease (VHL) is an autosomal dominant disorder, caused by mutations of the VHL... more Von Hippel-Lindau disease (VHL) is an autosomal dominant disorder, caused by mutations of the VHL gene showing a strong genotype-phenotype correlation. The present report concerns a 16-year-old girl with VHL (retinal, spinal cord and cerebellar haemangioblastomas and pancreatic cysts), her father (retinal and spinal cord haemangioblastomas) and the phenotypically healthy mother and younger brother and sister. DNA extraction, PCR and direct sequencing of the VHL entire coding and intronic flanking sequences, were performed according to standard procedures. In the index patient and her father a novel heterozygous germline was identified; nonsense mutation (p.145X) in exon 2 of VHL, leading to a truncated VHL protein lacking the last 66 amino acids. This is the first report of a novel VHL mutation in patients with VHL associated with haemangioblastomas and pancreatic cysts but not renal cell carcinoma.

Nature Genetics, 2006

Phosphodiesterases (PDEs) regulate cyclic nucleotide levels. Increased cyclic AMP (cAMP) signalin... more Phosphodiesterases (PDEs) regulate cyclic nucleotide levels. Increased cyclic AMP (cAMP) signaling has been associated with PRKAR1A or GNAS mutations and leads to adrenocortical tumors and Cushing syndrome 1-7 . We investigated the genetic source of Cushing syndrome in individuals with adrenocortical hyperplasia that was not caused by known defects. We performed genome-wide SNP genotyping, including the adrenocortical tumor DNA. The region with the highest probability to harbor a susceptibility gene by loss of heterozygosity (LOH) and other analyses was 2q31-2q35. We identified mutations disrupting the expression of the PDE11A isoform-4 gene (PDE11A) in three kindreds. Tumor tissues showed 2q31-2q35 LOH, decreased protein expression and high cyclic nucleotide levels and cAMP-responsive element binding protein (CREB) phosphorylation. PDE11A codes for a dual-specificity PDE that is expressed in adrenal cortex and is partially inhibited by tadalafil and other PDE inhibitors 8,9 ; its germline inactivation is associated with adrenocortical hyperplasia, suggesting another means by which dysregulation of cAMP signaling causes endocrine tumors.

Molecular Endocrinology, 2005

binding. In the present work, we functionally and structurally characterized this CRAC motif usin... more binding. In the present work, we functionally and structurally characterized this CRAC motif using reconstituted recombinant PBR and nuclear magnetic resonance. Deletion of the C-terminal domain of PBR and mutation of the highly conserved among all PBR amino acid sequences Y152 of the CRAC domain resulted in loss of the ability of mutant recPBR to bind cholesterol. Nuclear magnetic resonance analysis of a PBR C-terminal peptide (144-169) containing the CRAC domain indicated a helical conformation for the L144-S159 fragment. As a result of the side-chain distribution, a groove that could fit a cholesterol molecule is delineated, on one hand, by Y152, T148, and L144, and, on the other hand, by Y153, M149, and A145. The aromatic rings of Y152 and Y153 assigned as essential residues for cholesterol binding constitute the gate of the groove. Furthermore, the side chain of R156 may cap the groove by interacting with the sterol hydroxyl group. These results provide structural and functional evidence supporting the finding that the CRAC domain in the cytosolic carboxyl-terminal domain of PBR might be responsible for the uptake and translocation of cholesterol into the mitochondria. (Molecular Endocrinology 19: 588-594, 2005)