Death-associated protein 3 is overexpressed in human thyroid oncocytic tumours.

BACKGROUND: The human death-associated protein 3 (hDAP3) is a GTP-binding constituent of the small subunit of the mitochondrial ribosome with a pro-apoptotic function. METHODS: A search through publicly available microarray data sets showed 337 genes potentially coregulated with the DAP3 gene. The promoter sequences of these 337 genes and 70 out of 85 mitochondrial ribosome genes were analysed in silico with the DAP3 gene promoter sequence. The mitochondrial role of DAP3 was also investigated in the thyroid tumours presenting various mitochondrial contents. RESULTS: The study revealed nine transcription factors presenting enriched motifs for these gene promoters, five of which are implicated in cellular growth (ELK1, ELK4, RUNX1, HOX11-CTF1, TAL1-ternary complex factor 3) and four in mitochondrial biogenesis (nuclear respiratory factor-1 (NRF-1), GABPA, PPARG-RXRA and estrogen-related receptor alpha (ESRRA)). An independent microarray data set showed the overexpression of ELK1, RUNX1 and ESRRA in the thyroid oncocytic tumours. Exploring the thyroid tumours, we found that DAP3 mRNA and protein expression is upregulated in tumours presenting a mitochondrial biogenesis compared with the normal tissue. ELK1 and ESRRA were also showed upregulated with DAP3. CONCLUSION: ELK1 and ESRRA may be considered as potential regulators of the DAP3 gene expression. DAP3 may participate in mitochondrial maintenance and play a role in the balance between mitochondrial homoeostasis and tumourigenesis.

[Energy metabolism of the cancer cell: example of mitochondria-rich endocrine tumors]. / Métabolisme énergétique de la cellule cancéreuse: exemple des tumeurs endocrines riches en mitochondries. Conférence donnée lors de la réunion annuelle du GTE Angers--17 novembre 2005.

Most solid tumours preferentially develop glycolytic metabolism, often accompanying tumor aggressiveness. Increase in nucleic acid synthesis is associated with cell proliferation and glucose shunting to the pentose phosphate pathway. High glucose consumption is more associated with a metabolic adaptation than with a mitochondrial defect. Tumor cells do not present specific genetic modifications but adapt their metabolic capacities to their priority needs. However their metabolisms depend on oncogene expression more specifically expressed in this context. The glycolytic pathway is favored by tumor proliferation under hypoxia. Stabilization of HIF1 factor may explain the glycolytic metabolism of the tumors in an anaerobic environment. We demonstrate in two types of mitochondrial rich tumors, that specific defects induce completely different metabolic directions: when familial paragangliomas present a glycolytic metabolism, thyroid oncocytic tumors develop a specific oxidative metabolism.

Relationships between angiotensin I converting enzyme gene polymorphism, plasma levels, and diabetic retinal and renal complications.

Insulin-dependent diabetes mellitus (IDDM), cardiovascular morbidity, and vital prognosis are linked to diabetic nephropathy, which is probably determined by renal hemodynamic abnormalities and by a genetic predisposition. Angiotensin I converting enzyme (ACE) regulates systemic and renal circulations through angiotensin II formation and kinins metabolism. Plasma and cellular ACE levels are genetically determined; an insertion/deletion polymorphism of the ACE gene is strongly associated with ACE levels, subjects homozygote for insertion (genotype II) having the lowest plasma values. We studied the relationship between the ACE gene polymorphism or plasma levels and microcirculatory disorders of IDDM through two independent studies: one involved 57 subjects with or without diabetic retinopathy, and the other compared 62 IDDM subjects with diabetic nephropathy to 62 diabetic control subjects with the same characteristics (including retinopathy severity) but with normal kidney function. The ACE genotype distribution was not different in diabetic subjects with or without retinopathy and in a healthy population. Conversely, an imbalance of ACE genotype distribution, with a low proportion of II subjects, was observed in IDDM subjects with diabetic nephropathy compared with their control subjects (P = 0.006). Plasma ACE levels were mildly elevated in all diabetic groups, independently of retinopathy, but they were higher in subjects with nephropathy than in those without nephropathy (P = 0.0022). The II genotype of ACE gene is a marker for reduced risk for diabetic nephropathy.

Defective mitochondrial ATP synthesis in oxyphilic thyroid tumors.

Oxyphilic tumors (oncocytomas or Hürthle cell tumors) form a rare subgroup of thyroid tumors characterized by cells containing abundant mitochondria. The relationship between the mitochondrial proliferation and the pathogenesis of these tumors is unknown. We have assessed the expression of the mitochondrial ND2 and ND5 (subunits of the nicotinamide adenine dinucleotide dehydrogenase complex) genes and the nuclear UCP2 (uncoupling protein 2) gene in 22 oxyphilic thyroid tumors and matched controls. The consumption of oxygen in mitochondria from tumors was determined by polarography. ATP assays were used to explore the mitochondrial respiratory chain activity and the oxidative phosphorylation coupling in seven fresh thyroid tumors and controls. Adenosine triphosphate synthesis was significantly lower in all the tumors, compared with controls, suggesting that a coupling defect in oxidative phosphorylation may be a cause of mitochondrial hyperplasia in oxyphilic thyroid tumors.

Analysis of Tg transcripts by real-time RT-PCR in the blood of thyroid cancer patients.

Serum Tg (sTg) assays are sometimes unsatisfactory for monitoring thyroid cancer because interference caused by anti-Tg antibodies may reduce the sensitivity of the tests during thyroid hormone therapy. We have therefore developed a complementary method using real-time quantitative RT-PCR based on the amplification of Tg mRNA. Two different pairs of primers were used for the determination of the frequency of one of the variants of the alternative splicing of Tg mRNA. The frequency of this variant was as high in patients (n = 40) as in controls (n = 30), accounting for about 33% of the total Tg mRNA. Using appropriate primers, we observed that Tg mRNA values in controls varied according to the volume of thyroid tissue and the TSH concentration. The Tg mRNA values allowed the definition of a positive cutoff point at 1 pg/microg total RNA. This cutoff point, tested on the group of patients treated for thyroid cancer, produced fewer false negative results than those obtained with sTg assays. The standardized, highly sensitive real-time RT-PCR technique may therefore prove useful as a complement to sTg assays, particularly for patients with recurrent thyroid cancer receiving T(4) therapy.

Familial medullary thyroid carcinoma with noncysteine ret mutations: phenotype-genotype relationship in a large series of patients.

Familial medullary thyroid carcinoma only is related to germline mutations in the protooncogene RET, mainly in exons 10, whereas noncysteine mutations (exons 13-15) are considered infrequent. We analyzed 148 patients from 47 familial medullary thyroid carcinoma only families, and we found noncysteine RET mutations in 59.5% of these families. Of the index cases with noncysteine mutations, 43.4% presented with a multinodular goiter and high basal calcitonin; they were older at diagnosis than those with mutation in exon 10 and had more multifocal medullary thyroid carcinoma, but no difference in size, bilaterality, presence of C cell hyperplasia, or nodal metastases was found. Gene carriers with noncysteine RET mutations had a lower incidence of medullary thyroid carcinoma (78.2% vs. 94.1%) than those with mutation in exon 10; 20.2% had C cell hyperplasia only, although thyroidectomized at an older age. In conclusion, familial medullary thyroid carcinoma with noncysteine RET mutations are not infrequent and are overrepresented in presumed sporadic medullary thyroid carcinoma, suggesting that RET analysis should routinely be extended to exons 13, 14, and 15. The phenotype is characterized by a late onset of the disease, suggesting a delayed appearance of C cell disease rather than a less aggressive form. In familial medullary thyroid carcinoma gene carriers, the optimal timing for thyroidectomy remains controversial. Based on these data, we propose that surgery should be performed before elevation of the basal calcitonin level, potentially as soon as the pentagastrin test becomes abnormal.

mtDNA haplogroup J: a contributing factor of optic neuritis.

Optic neuritis frequently occurs in multiple sclerosis (MS), and shares several similarities with the optic neuritis of Leber's hereditary optic neuropathy (LHON), which is mainly due to maternally transmitted mitochondrial DNA (mtDNA) mutations. Our report shows for the first time that a mitochondrial DNA background could influence the clinical expression of MS. One European mtDNA haplogroup was found only in MS patients with optic neuritis but not in MS patients without visual symptoms. Therefore, we hypothesize that mtDNA haplogroup J might constitute a risk factor for optic neuritis occurrence when it is coincidentally associated with MS, but not be a risk factor for developing MS per se as suggested previously.

Mitochondrial activity in XTC.UC1 cells derived from thyroid oncocytoma.

Thyroid oncocytoma is characterized by the presence of oncocytes containing abnormally large numbers of mitochondria. However, the relationship between the abundance of mitochondria and the pathogenesis of the tumors is unknown. Recently, a new cell line, named XTC.UC1, has been derived from a metastasis of thyroid oncocytoma. We have studied the metabolism and the gene expression profile of the mitochondria in XTC.UC1 cells, using B-CPAP cells as controls. There were no signs of mitochondrial respiratory chain defects or uncoupling between the respiratory chain and adenosine triphosphate (ATP) production. In XTC.UC1 cells, mtDNA transcripts were increased more than fivefold than in controls, in parallel with a 3.6-fold increase in mtDNA content. Finally, in spite of the glycolytic metabolism induced by the culture medium, the mitochondria of XTC.UC1 cells possess the phenotype of oncocytic cells with hypertrophic mitochondria, higher respiratory enzyme activity and higher mtDNA content than in controls. XTC.UC1 cells may therefore offer a useful model for investigating the coordination of the nuclear and mitochondrial genomes, in the context of thyroid tumors.

No evidence of thyrotropin receptor and G(s alpha) gene mutation in high iodine uptake thyroid carcinoma.

Usually, thyroid carcinoma presents as a cold nodule on radioiodine scintigraphy. High-uptake nodules on iodine thyroid scans are associated with an exceedingly low incidence of malignancy. Only 29 cases of carcinomas appearing as hot or warm nodules have as yet been reported. From 1993 to 1999, we have observed eight similar cases (4 hot and 4 warm thyroid nodules) suggesting that thyroid carcinomas may not be as rare as usually considered in these circumstances. Four tumors were available for molecular analysis on paraffin-embedded sections. Because no mutations were found in the whole coding portions of thyrotropin-receptor (TSH-R) gene and fragments encompassing the mutational hot spots of the G(s alpha) gene, it is unlikely that activating mutations of the TSH-R or G(s alpha) genes were involved in these carcinomas.

Microarray analysis refines classification of non-medullary thyroid tumours of uncertain malignancy.

Conventional histology failed to classify part of non-medullary thyroid lesions as either benign or malignant. The group of tumours of uncertain malignancy (T-UM) concerns either atypical follicular adenomas or the recently called 'tumours of uncertain malignant potential'. To refine this classification we analysed microarray data from 93 follicular thyroid tumours: 10 T-UM, 3 follicular carcinomas, 13 papillary thyroid carcinomas and 67 follicular adenomas, compared to 73 control thyroid tissue samples. The diagnosis potential of 16 selected genes was validated by real-time quantitative RT-PCR on 6 additional T-UM. The gene expression profiles in several groups were examined with reference to the mutational status of the RET/PTC, BRAF and RAS genes. A pathological score (histological and immunohistochemical) was estimate for each of the T-UM involved in the study. The correlation between the T-UM gene profiles and the pathological score allowed a separation of the samples in two groups of benign or malignant tumours. Our analysis confirms the heterogeneity of T-UM and highlighted the molecular similarities between some cases and true carcinomas. We demonstrated the ability of few marker genes to serve as diagnosis tools and the need of a T-UM pathological scoring.

The status of CDKN2A alpha (p16INK4A) and beta (p14ARF) transcripts in thyroid tumour progression.

CDKN2A locus on chromosome 9p21 encodes two tumour suppressor proteins pl6INK4A, which is a regulator of the retinoblastoma (RB) protein, and p14ARF, which is involved in the ARF-Mdm2-p53 pathway. The aim of this study was to determine if CDKN2A gene products are implicated in differentiated thyroid carcinogenesis and progression. We used real-time quantitative RT-PCR and immunohistochemistry to assess both transcripts and proteins levels in 60 tumours specimens. Overexpression of p14ARF and pl6INK4A was observed in follicular adenomas, follicular carcinomas and papillary carcinomas, while downregulation was found in oncocytic adenomas compared to nontumoral paired thyroid tissues. These deregulations were statistically significant for pl6INK4a (P=0.006) in follicular adenomas and close to statistical significance for p14ARF in follicular adenomas (P=0.06) and in papillary carcinomas (P=0.05). In all histological types, except papillary carcinomas, we observed a statistically significant relationship between p14ARF and E2F1 (r=0.64 to 1, P<0.05). Our data are consistent with involvement of CDKN2A transcript upregulation in thyroid follicular tumorigenesis as an early event. However, these deregulations do not appear to be correlated to the clinical outcome and they could not be used as potential prognostic markers.

Long PCR analysis of human gamete mtDNA suggests defective mitochondrial maintenance in spermatozoa and supports the bottleneck theory for oocytes.

The long PCR and the Southern blot techniques were used to study mitochondrial DNA (mtDNA) in 94 sperm samples, and in 35 oocytes collected from 12 women. The sperm samples were classified in two sets: 37 samples from normal subjects, and 57 samples from patients with oligoasthenospermia. In both sets, most of the spermatozoan mitochondria had multiple mtDNA deletions. The rate of mtDNA mutation, which appears unexpectedly high, considering the short life span of the spermatozoa, may be due to impaired maintenance during differentiation. In contrast, despite the long life span of oocytes and the extended meiotic period, oocyte mitochondria showed few mtDNA rearrangements. However, mitochondria in oocytes from a given donor revealed considerable mutational heterogeneity. This supports the bottleneck theory of rapid segregation of mtDNA genotypes during early oogenesis. The long PCR technique, which allows analysis of the entire mitochondrial genome, provides new information on mtDNA instability in human gametes. Our findings suggest that mtDNA maintenance differs in the two types of gametes.

A conserved N-terminal sequence targets human DAP3 to mitochondria.

Human DAP3 (death-associated protein-3) has been identified as an essential positive mediator of programmed cell death. Structure-function studies have shown previously the N-terminal extremity of the protein to be required in apoptosis induction. Analysis of human DAP3 gene structure predicted 13 exons and subsequent targeting prediction by two software packages (MITOPROT and TargetP) gave a high probability for mitochondrial targeting. The predicted N-terminal targeting structure was also found in the mouse, Drosophila, and C. elegans orthologues with a strong sequence homology between mouse and human. Secondary structure analyses identified alpha-helical structures typical of mitochondrial target peptides. To confirm experimentally this targeting we constructed a fusion protein with N-terminal human DAP3 upstream of enhanced green fluorescent protein (EGFP). Confocal analysis of transfected human fibroblasts clearly demonstrated EGFP localization exclusive to mitochondria. The positioning of this key apoptotic factor at the heart of the mitochondrial pathway provides exciting insight into its role in programmed cell death.

Increased sperm mitochondrial DNA content in male infertility.

BACKGROUND: There is increasing evidence that mitochondrial DNA (mtDNA) anomalies in sperm may lead to infertility. Point mutations, deletions and the presence of a specific mtDNA haplogroup have been associated with poor sperm quality, but little attention has been paid to the role of mtDNA content. METHODS: Using density gradient separation and swim-up methods, we selected motile sperm from 32 normal and 35 abnormal sperm samples. The mtDNA/beta-globin gene ratio was determined by real-time quantitative PCR. RESULTS: The average mtDNA/beta-globin ratio of sperm collected from 100% density layers was 1.4 for normal sperm, 6.1 for sperm samples presenting at least one abnormal criterion [among the three criteria established by World Health Organization (1999), i.e. sperm count, motility and morphology], and 9.1 for sperm samples presenting two or more of these abnormal criteria. These differences are very highly significant (P < 0.0001). The mtDNA numbers were also much greater in sperm collected from the 40% density gradient layers (mean: 17.1, P < 0.001), known to contain the most abnormal sperm of the sperm samples, than in those collected from the 100% layers known to contain sperm with the best fertilizing ability. CONCLUSION: Our results showed significant mtDNA amplification in sperm collected from abnormal sperm samples.

Mitochondrial DNA content affects the fertilizability of human oocytes.

Mitochondrial DNA content varies considerably in oocytes, even when collected from the same patient. In the present study, real-time quantitative polymerase chain reaction analysis of 113 unfertilized oocytes obtained from 43 patients revealed an average of 193,000 (range: 20,000 to 598,000) mitochondrial genomes per cell. We compared several groups of oocytes to investigate the relationship between mitochondrial DNA content and fertilizability. The average mitochondrial DNA copy number was significantly lower in cohorts suffering from fertilization failure compared to cohorts with a normal rate of fertilization. In addition, the mitochondrial copy number of oocytes from patients with fertilization failure due to unknown causes was significantly lower than that of oocytes from patients in which IVF failure was due mainly to a severe sperm defect. The lower mtDNA copy number could be due to defective cytoplasmic maturation of oocytes. We conclude that low mitochondrial DNA content, due to inadequate mitochondrial biogenesis or cytoplasmic maturation, may adversely affect oocyte fertilizability.