Jean-Michel Bidart

ROS-generating NADPH oxidase NOX4 is a critical mediator in oncogenic H-Ras-induced DNA damage and subsequent senescence

Activated Ras oncogene induces DNA-damage response by triggering reactive oxygen species (ROS) production and this is critical for oncogene-induced senescence. Until now, little connections between oncogene expression, ROS-generating NADPH oxidases and DNA-damage response have emerged from different studies. Here we report that H-RasV12 positively regulates the NADPH oxidase system NOX4-p22phox that produces H2O2. Knocking down the NADPH oxidase with small interference RNA decreases H-RasV12-induced DNA-damage response detected by γ-H2A.X foci analysis. Using HyPer, a specific probe for H2O2, we detected an increase in H2O2 in the nucleus correlated with NOX4-p22phox perinuclear localization. DNA damage response can be caused not only by H-RasV12-driven accumulation of ROS but also by a replicative stress due to a sustained oncogenic signal. Interestingly, NOX4 downregulation by siRNA abrogated H-RasV12 regulation of CDC6 expression, an essential regulator of DNA replication. Moreover, senescence markers, such as senescence-associated heterochromatin foci, PML bodies, HP1β foci and p21 expression, induced under H-RasV12 activation were decreased with NOX4 inactivation. Taken together, our data indicate that NADPH oxidase NOX4 is a critical mediator in oncogenic H-RasV12-induced DNA-damage response and subsequent senescence.

Elevation of CA 125 in patients with benign and malignant ascites

The presence of CA 125, an ovarian cancer‐associated antigen, was assessed in serum and ascites from patients with ovarian cancer (n = 47), hepatocellular carcinoma (n = 21), and liver cirrhosis (n = 40). Abnormal levels of serum CA 125 were observed in 49% of ovarian cancer patients, and in 89% of these same patients with ascites. In all cases of cirrhosis or hepatocellular carcinoma with ascites, CA 125 levels were above 35 U/ml. The specificity and sensitivity of CA 125 measurement for detecting ascites in chronic liver diseases were 73% and 100%, respectively. Furthermore, the CA 125 level was always higher in ascites than in serum. The authors conclude that CA 125 is a nonspecific marker of ascites. This result must be considered in the interpretation of CA 125 elevation in patients with ovarian cancer. Moreover, CA 125 may be of value in the diagnosis and monitoring of peritoneal diseases. Cancer 59:213–217, 1987.

Canstatin Acts on Endothelial and Tumor Cells via Mitochondrial Damage Initiated through Interaction with αvβ3 and αvβ5 Integrins

Canstatin, the noncollagenous domain of collagen type IV α-chains, belongs to a series of collagen-derived angiogenic inhibitors. We have elucidated the functional receptors and intracellular signaling induced by canstatin that explain its strong antitumor efficacy in vivo . For this purpose, we generated a canstatin-human serum albumin (CanHSA) fusion protein, employing the HSA moiety as an expression tag. We show that CanHSA triggers a crucial mitochondrial apoptotic mechanism through procaspase-9 cleavage in both endothelial and tumor cells, which is mediated through cross-talk between α v β 3 - and α v β 5 -integrin receptors. As a point of reference, we employed the first three kringle domains of angiostatin (K1-3), fused with HSA, which, in contrast to CanHSA, act only on endothelial cells through α v β 3 -integrin receptor–mediated activation of caspase-8 alone, without ensuing mitochondrial damage. Taken together, these results provide insights into how canstatin might exert its strong anticancer effect.

Medullary thyroid carcinoma as part of a multiple endocrine neoplasia type 2B syndrome: influence of the stage on the clinical course.

Multiple endocrine neoplasia type 2B (MEN 2B) is an exceptional syndrome, for which the optimal age of thyroidectomy is poorly established and the course of medullary thyroid carcinoma (MTC) is ill‐defined.

Differences in Recognition of the 1st WHO International Reference Reagents for hCG-Related Isoforms by Diagnostic Immunoassays for Human Chorionic Gonadotropin

BACKGROUND The 1st WHO International Reference Reagents (IRRs) for 6 human chorionic gonadotropin (hCG)-related molecular variants, highly purified and calibrated in substance concentrations by the IFCC Working Group for hCG, permit experimental elucidation of what commercially available hCG methods measure in molar terms and enable assessment of their fitness for clinical purposes. METHODS Pools containing known amounts of the IRRs spiked into normal human serum were issued to participants through the UK National External Quality Assessment Service for hCG for a period of 7 years. Among 16 assays used, 4 recognized only hCG, whereas 6 recognized hCG and its free beta-subunit (hCGbeta), and 6 recognized hCG, hCGbeta, and the beta core fragment. RESULTS Differences in calibration of current hCG assays are moderate. Mean recovery of the current International Standard (IS), hCG IS 75/589, was 107% (range 93% to 126%), whereas that of the IRR 99/688 for hCG was 139% (range 109%-164%). Between-method variation for the latter (CV 12.3%) was also greater than for IS 75/589 (CV 8.8%). Recognition of hCGbeta varied markedly (CV 37%). Most assays overestimated it, but 2 RIAs produced results that were slight underestimations. Recognition of the beta core fragment was even more variable (CV 57%) and was closest to equimolarity for the RIAs. CONCLUSIONS Assays for hCG show considerable variation in their recognition of various forms of hCG, and this variability is the most important cause of method-related differences in hCG results in serum and an even more important cause of method-related differences in urine measurements. Equimolar recognition of the major hCG isoforms is essential if between-method comparability for hCG is to be improved.

Follicular Thyroid Tumors with the PAX8-PPARγ1 Rearrangement Display Characteristic Genetic Alterations

Follicular thyroid carcinomas (FTC) arise through oncogenic pathways distinct from those involved in the papillary histotype. Recently, a t(2;3)(q13;p25) rearrangement, which juxtaposes the thyroid transcription factor PAX8 to the peroxisome proliferator-activated receptor (PPAR) gamma1, was described in FTCs. In this report, we describe gene expression in 11 normal tissues, 4 adenomas, and 8 FTCs, with or without the PAX8-PPARgamma1 translocation, using custom 60-mer oligonucleotide microarrays. Results were confirmed by quantitative real-time polymerase chain reaction of 65 thyroid tissues and by immunohistochemistry. Statistical analysis revealed a pattern of 93 genes discriminating FTCs, with or without the translocation, that were morphologically undistinguishable. Although the expression of thyroid-specific genes was detectable, none appeared to be differentially regulated between tumors with or without the translocation. Differentially expressed genes included genes related to lipid/glucose/amino acid metabolism, tumorigenesis, and angiogenesis. Surprisingly, several PPARgamma target genes were up-regulated in PAX8-PPARgamma-positive FTCs such as angiopoietin-like 4 and aquaporin 7. Moreover many genes involved in PAX8-PPARgamma expression profile presented a putative PPARgamma-promoter site, compatible with a direct activity of the fusion product. These data identify several differentially expressed genes, such as FGD3, that may serve as potential targets of PPARgamma and as members of novel molecular pathways involved in the development of thyroid carcinomas.

Radiation and inhibition of angiogenesis by canstatin synergize to induce HIF-1α–mediated tumor apoptotic switch

Tumor radioresponsiveness depends on endothelial cell death, which leads in turn to tumor hypoxia. Radiation-induced hypoxia was recently shown to trigger tumor radioresistance by activating angiogenesis through hypoxia-inducible factor 1-regulated (HIF-1-regulated) cytokines. We show here that combining targeted radioiodide therapy with angiogenic inhibitors, such as canstatin, enhances direct tumor cell apoptosis, thereby overcoming radio-induced HIF-1-dependent tumor survival pathways in vitro and in vivo. We found that following dual therapy, HIF-1alpha increases the activity of the canstatin-induced alpha(v)beta(5) signaling tumor apoptotic pathway and concomitantly abrogates mitotic checkpoint and tetraploidy triggered by radiation. Apoptosis in conjunction with mitotic catastrophe leads to lethal tumor damage. We discovered that HIF-1 displays a radiosensitizing activity that is highly dependent on treatment modalities by regulating key apoptotic molecular pathways. Our findings therefore support a crucial role for angiogenesis inhibitors in shifting the fate of radiation-induced HIF-1alpha activity from hypoxia-induced tumor radioresistance to hypoxia-induced tumor apoptosis. This study provides a basis for developing new biology-based clinically relevant strategies to improve the efficacy of radiation oncology, using HIF-1 as an ally for cancer therapy.

Expression of nicotinamide adenine dinucleotide phosphate oxidase flavoprotein DUOX genes and proteins in human papillary and follicular thyroid carcinomas.

Duox2, and probably Duox1 are glycoflavoproteins involved in the thyroid H2O2 generator functionally associated to thyroperoxidase (TPO). We investigated both DUOX1 and DUOX2 gene expressions using quantitative reverse transcription-polymerase chain reaction (RT-PCR) in 47 thyroid carcinomas, including 10 paired normal/tumoral tissues. In carcinomas, variations of DUOX1 and DUOX2 mRNA levels were parallel, indicating that control mechanisms of both gene expressions operate in tumors as well as in normal thyroid tissues; DUOX1 expression was in the normal range in 20, was decreased up to 50-fold in 8, and increased up to 7-fold in 19 samples. DUOX2 expression was in the normal range in 15, was decreased up to 200-fold in 10, and increased up to 5-fold in 22 samples. In the 10 paired samples, variations of DUOX and TPO gene expressions were not correlated. We analyzed Duoxl/2 protein expression in 86 tumor samples using an antipeptide antiserum reacting with both Duox proteins. In normal tissue, Duox proteins are localized at the apical pole of thyrocytes, with 40% to 60% of thyrocytes being stained. In the 86 cancer tissues, immunostaining was absent in 19 samples, was low in 32, and normal or even slightly increased in the other 35 samples. The expression of Duox proteins was related to tumor differentiation, being more frequently found in neoplastic tissues that were able to pick up radioiodine, and in those with a detectable expression of sodium iodide symporter (NIS), pendrin and TPO.

Chromogranin A and the α -subunit of glycoprotein hormones in medullary thyroid carcinoma and phaeochromocytoma

Using specific immunoradiometric assays, we evaluated the clinical usefulness of chromogranin A and the α-subunit of glycoprotein hormones in neuroendocrine tumours of neuroectodermic origin. The serum α-subunit of glycoprotein hormones was only slightly increased in 2 out of 44 medullary thyroid carcinoma or phaeochromocytoma patients with increased calcitonin or 24-hour urinary metanephrine levels. Serum chromogranin A was increased in 12 of 45 (27%) medullary thyroid carcinoma patients with an elevated calcitonin level and in 4 of 16 medullary thyroid carcinoma patients (25%) with an undetectable calcitonin level, in 5 of 7 phaeochromocytoma patients with increased urinary catecholamine and metabolite excretion, and in 2 of 3 patients with a non-functioning phaeochromocytoma. During follow-up, the course of chromogranin A was found to parallel that of tumour burden and/or 24-hour urinary metanephrine in 5 phaeochromocytoma patients. We conclude that chromogranin A measurement is not recommended for the diagnosis of medullary thyroid carcinoma patients. It may be useful in patients with functioning and non-functioning phaeochromocytomas as a follow-up marker. In neuroendocrine tumour patients with elevated calcitonin secretion, the serum α-subunit of glycoprotein hormone measurement may help differentiate medullary thyroid carcinoma or phaeochromocytoma patients from other endodermal-derived neuroendocrine tumour patients in whom it is frequently elevated.

Standardization Of Protein Immunoprocedures Choriogonadotropin (Cg)

The objectives of this report is to improve the quality of immunochemical determinations of proteins. Procedures for determination of the various molecular forms hCG were chosen as the subject of a detailed study, with the aim as using this report as a model for similar future projects. The aim of this undertaking is to improve the reliability and reproducibility of the results and, as a result of this, to enhance the clinical usefulness of hCG determinations. The following means will be used to achieve these goals: (a) establishing uniform nomenclature and abbreviations for various molecular forms of hCG, (b) preparation of new calibrators for these and establishing of methods for determining the amount of substance concentration in mol/L, (c) improving quality assessment materials and procedures, (d) definition of methods for characterization of hCG measurement procedures.