Imaging of the vulnerable carotid plaque: biological targeting of inflammation in atherosclerosis using iron oxide particles and MRI.

OBJECTIVES: Identification of those patients with high-risk asymptomatic carotid plaques remains an elusive but essential step in stroke prevention. Inflammation is a key process in plaque destabilization and the propensity of atherosclerotic lesions to cause clinical sequelae. There is currently no clinical imaging technique available to assess the degree of inflammation associated with plaques. This study aims at visualizing and characterizing atherosclerosis using antibody-conjugated superparamagnetic iron oxide (SPIO) particles as an MRI probe to assess inflammation in human atherosclerotic plaques. METHODS: Atherosclerotic plaques were collected from 20 consecutive patients (n=10 from symptomatic patients, n=10 from asymptomatic patients) undergoing carotid endarterectomy (CEA) for extracranial high-grade internal carotid artery (ICA) stenosis (>70% luminal narrowing). Inflammatory markers on human atherosclerotic plaques were detected and characterized by ex vivo magnetic resonance imaging (MRI) using anti-VCAM-1 antibody and anti-E-selectin antibody-conjugated SPIO with confirmatory immunohistochemistry. RESULTS: Inflammation associated with human ex vivo atherosclerotic plaques could be imaged using dual antibody-conjugated SPIO by MRI. Symptomatic plaques could be distinguished from asymptomatic ones by the degree of inflammation, and the MR contrast effect was significantly correlated with the degree of plaque inflammation (r=.64, p<.001). The asymptomatic plaque population exhibited heterogeneity in terms of inflammation. The dual-targeted SPIO-induced MR signal not only tracked closely with endothelial activation (i.e. endothelial expression of VCAM-1 and E-selectin), but also reflected the macrophage burden within plaque lesions, offering a potential imaging tool for quantitative MRI of inflammatory activity in atherosclerosis. CONCLUSIONS: These functional molecular MRI probes constitute a novel imaging tool for ex vivo characterization of atherosclerosis at a molecular level. Further development and translation into the clinical arena will facilitate more accurate risk stratification in carotid artery disease in the future.

Cardiometabolic and immune factors associated with increased common carotid artery intima-media thickness and cardiovascular disease in patients with systemic lupus erythematosus.

BACKGROUND AND AIM: Patients with systemic lupus erythematosus (SLE) have a higher prevalence of subclinical atherosclerosis and higher risk of cardiovascular (CV) events compared to the general population. The relative contribution of CV-, immune- and disease-related risk factors to accelerated atherogenesis in SLE is unclear. METHODS AND RESULTS: Fifty SLE patients with long-lasting disease (mean age 44 ± 10 years, 86% female) and 50 sex- and age-matched control subjects were studied. Common carotid artery intima-media thickness (CCA-IMT) was used as a surrogate marker of atherosclerosis. We evaluated traditional and immune- and disease-related factors, assessed multiple T-cell subsets by 10-parameter-eight-colour polychromatic flow cytometry and addressed the effect of pharmacological therapies on CCA-IMT. In SLE patients, among several cardiometabolic risk factors, only high-density lipoprotein levels (HDL) and their adenosine triphosphate-binding cassette transporter 1 (ABCA-1)-dependent cholesterol efflux capacity were markedly reduced (p < 0.01), whereas the CCA-IMT was significantly increased (p = 0.03) compared to controls. CCA-IMT correlated with systolic blood pressure, low-density lipoprotein (LDL) cholesterol and body mass index (BMI), but not with disease activity and duration. The activated CD4(+)HLA-DR(+) and CCR5(+) T-cell subsets were expanded in SLE patients. Patients under hydroxychloroquine (HCQ) therapy showed lower CCA-IMT (0.62 ± 0.08 vs. 0.68 ± 0.10 mm; p = 0.03) and better risk-factor profile and presented reduced circulating pro-atherogenic effector memory T-cell subsets and a parallel increased percentage of naïve T-cell subsets. CONCLUSION: HDL represents the main metabolic parameter altered in SLE patients. The increased CCA-IMT in SLE patients may represent the net result of a process in which 'classic' CV risk factors give a continuous contribution, together with immunological factors (CD4(+)HLA-DR(+) T cells) which, on the contrary, could contribute through flares of activity of various degrees over time. Patients under HCQ therapy present a modified metabolic profile, a reduced T-cell activation associated with decreased subclinical atherosclerosis.

Local gentamicin-collagen sponge reduces cardiovascular implantable electronic device infections and pocket hematoma.

OBJECTIVE: Implantation or replacement of a cardiovascular implantable electronic device (CIED) may be associated with complications, such as pocket hematomas and infections. This study aims to determine whether a lyophilized gentamycin-containing collagen implant (GCCI) reduces major CIED infections and pocket hematomas after implant. SUBJECTS AND METHODS: A retrospective study was conducted among patients who underwent implantation or replacement of CIED at the Tor Vergata Polyclinic (Rome, Italy) between June 2007 and November 2019. The primary combined endpoint was infection and hematoma occurrence through 12 months of follow-up post-procedure. The rate of single infectious complications, pocket hematomas or both were also assessed. RESULTS: We compared 475 patients treated with the GCCI (GCCI group) with 714 patients who did not receive it (control group). Complications occurred in 127 patients (11%); a statistically significant reduction of infections and pocket hematomas in the GCCI group was reported when compared with control patients (1% vs. 17%; p<0.0001). A total of 20 (2%) infectious events were reported, 102 (8%) patients developed a pocket hematoma, and 5 (0.4%) had both. The rate of single complications was significantly lower in GCCI group: infection 0.2% vs. 2.6% (p=0.002), pocket hematoma 0.6% vs. 13.8% (p<0.001). The association between antiplatelet/anticoagulation therapy and hematoma development was not statistically significant. CONCLUSIONS: The GCCI is a medical device that can be used in addition to local hemostasis and prophylactic doses of systemic antibiotics with the aim of reducing infective complications and pocket hematoma after permanent CIED implantation or replacement.

The use of contrast enhanced ultrasound in carotid arterial disease.

Traditionally, stroke risk stratification has centred on the degree of internal carotid artery stenosis, and the presence of focal neurological symptoms. However, degree of stenosis alone is a relatively poor predictor of future stroke in asymptomatic patients; the Asymptomatic Carotid Surgery Trial highlighting the need to identify a subgroup of asymptomatics that may benefit from intervention. Attempting to define this subgroup has inspired imaging research to identify, in vivo, high-risk plaques. In addition to pre-operative risk stratification of carotid stenosis, contrast enhanced ultrasound (CEUS) may be employed in monitoring response to plaque-stabilising therapies. Unlike most contrast agents used for computed tomography and magnetic resonance imaging, microbubbles used in CEUS remain within the vascular space and can hence be used to study the vasculature. In addition to improving current carotid structural scans, CEUS has potential to add extra information on plaque characteristics. Furthermore, by targeting microbubbles to specific ligands expressed on vascular endothelium, CEUS may have the ability to probe plaque biology. This review describes the current carotid ultrasound examination and the need to improve it, rationale for imaging neovascularisation, use of CEUS to image neovascularisation, microbubbles in improving the structural imaging of plaque, potential problems with CEUS, and future directions.

Inhibitory effect of TIMP influences the morphology of varicose veins.

OBJECTIVES: Imbalance of matrix metalloproteinase enzymes (MMP) and their inhibitors (TIMPs) may contribute to the development of varicose veins. We hypothesised that, histological changes in varicose vein wall correlate with alterations in expression of MMP/TIMP. METHODS: Varicose veins (n=26) were compared with great saphenous vein (GSV) segments (n=11) from arterial bypass, and with arm and neck veins from fistula and carotid operations (n=13). Varicose vein wall thickness was measured, enabling categorisation as atrophic and hypertrophic. MMP-2, MT1-MMP, TIMP-2, and TIMP-3 expression were quantitatively analysed by immunohistochemistry. RESULTS: There was significantly higher expression of TIMP-2 (immunopositive area 4.34% versus 0.26%), linked with connective tissue accumulation in the tunica media of varicose veins as compared with arm and neck vein controls. TIMP-2 and TIMP-3 expression was higher in hypertrophic than atrophic segments (3.2% versus 0.99% for TIMP-2, 1.7% versus 0.08% for TIMP-3). Similarly, TIMP-2 and TIMP-3 had elevated expression in the thicker proximal varicose vein segments compared to distal (4.3% versus 1.3% for TIMP-2 and 0.94% versus 0.41% for TIMP-3). CONCLUSIONS: This study linked morphological changes in varicose vein walls with MMP/TIMP balance. A higher TIMP expression favours deposition of connective tissue and thus thicker vein wall, reducing matrix turnover by suppression of protease activity.

2D/3D accuracies of implant position after guided surgery using different surgical protocols: A retrospective study.

PURPOSE: To compare the 2D and 3D positional accuracy of four guided surgical protocols using an analysis of linear and angular deviations. METHODS: DICOM and .STLs files obtained from a CBCT and a digital impression were superimposed with software to plan implant position. Fifty-six patients were subdivided into 4 groups: FGA group (template support [Ts]: teeth [T]; bed preparation [Bp]: fully guided [FG]; implant insertion [Ii]: 3D template [3Dt]; device [D]: manual adapter [MA], FGM group (Ts: T; Bp: FG; Ii: 3Dt; D: fully guided mounter [FGM]), PG group (Ts: T; Bp: FG; Ii: manual; D: none) and MS group (Ts: mucosa; Bp: FG; Ii: 3Dt; D: FGM). The position of 120 implants was assessed by superimposing the planned and final position recorded with a digital impression. RESULTS: In FGA group, 3D deviations were 0.92 ± 0.52 mm at the implant head and 1.14 ± 0.54 mm at the apex, and the angular deviation (ang. dev.) was 2.45 ± 1.24°. In FGM group, were 0.911 ± 0.44 mm (head) and 1.11 ± 0.54 mm (apex), and the ang. dev. was 2.73 ± 1.96°. In PG group, were 0.95 ± 0.47 mm (head) and 1.17 ± 0.488 mm (apex), and the ang. dev. was 3.71 ± 1.67°. In MS group, were 1.15 ± 0.45 mm (head) and 1.42 ± 0.45 mm (apex), and the ang. dev. was 4.19 ± 2.62°. Ang. dev. of MS group was different from the other groups (P < 0.05). CONCLUSIONS: Guided surgery showed a sufficient accuracy.

In vitro 3D and gravimetric analysis of removed tooth structure for complete and partial preparations.

PURPOSE: To quantify and to compare a gravimetric and three-dimensional (3D) analysis of the removed tooth structure for different complete crown preparations. METHODS: A total of 80 molar resin teeth and 8 preparation finishing lines were chosen: 1 for metal ceramic crowns (MCC); 3 for zirconia all-ceramic crowns: knife edge (ZirKnE), chamfer (ZirCha), and shoulder (ZirSho); 4 for lithium disilicate: light chamfer (LDLCha), chamfer (LDCha), shoulder (LDSho) and table top. Teeth were individually weighed to high precision and then prepared following the preparation guidelines. The teeth were reweighed after preparation, and the amount of structural reduction was calculated. In addition, all teeth were scanned before and after preparation, and the 3D volume of removed dental tissue was calculated, superimposing the two .stl files, as a difference of the volumes before and after the preparation. Kruskal-Wallis statistical analysis was carried out to determine significant differences among the groups with a significance level of p<0.05. RESULTS: Both analyses showed that LDLCha, ZirKnE and table-top preparations produced the smallest amount of removed structure, whereas the preparations for MCC, ZirSho and LDSho were more destructive. For MCC, 2.6 times more tooth structure must be removed than for table top. ZirKnE was 17.82% and LDLCha was 21.51% more conservative than MCC. The data obtained through the volumetric method were similar with those obtained by gravimetric analysis. CONCLUSIONS: ZirKnE, LDLCha, and table-top preparations produced the least amount of tooth tissue removal. Three-dimensional volumetric analysis can be a possible alternative to gravimetric analysis.

Influence of preparation design and spacing parameters on the risk of chipping of crowns made with Cerec Bluecam before cementation.

PURPOSE: To evaluate the influence of the preparation design and spacing parameters on the risk of chipping of crowns made by CEREC Bluecam before cementation. METHODS: A knife-edge preparation and a chamfer preparation were made on upper premolars. The teeth were scanned and two Co-Cr alloy replicas were made. Fifteen full crowns were manufactured for four groups using CEREC. The groups differed in type of preparation (knife-edge (KE) or chamfer (CHA)) and spacing parameters: spacer (0 or 150µm), marginal adhesive gap (10 or 50 or 150µm) and margin thickness (0 or 300µm). The four groups were: CHA 150 (spacer)- 50 (marginal adhesive gap)- 0 (margin thickness), KE 150-50-0, KE 150-50-300 and KE 150-150-300. The crowns were loaded before cementation by using an Instron machine to simulate the masticatory load applied during a trial. Differences in means were compared using two-way ANOVA and a post-hoc test (Tukey Test). The level of significance was set at P=0.05. RESULTS: The fracture values, ordered from least to most resistant, were: KE 150-50-300 group, CHA 150-50-0 group, KE 150-50-0 group and KE 150-150-300 group. Two-way ANOVA revealed statistically significant differences between pairs of means (p<0.05). Tukey's test showed that restorations of the KE 150-150-300 group can withstand a load significantly higher than that of other groups (p<0.01). In this group, the failures were mostly minor chippings, while the other groups had mostly major chippings and fractures. CONCLUSIONS: Marginal adhesive gap can affect the trial of a full crown.

Different Methods for Inlay Production: Effect on Internal and Marginal Adaptation, Adjustment Time, and Contact Point.

The aim of this study was to evaluate the effect of different production methods of resin and ceramic inlays on marginal and internal adaptation, adjustment time, and proximal contacts. Forty premolars were selected, embedded (their roots), and prepared to receive inlays that were made as follows (n=10): LaRe-digital impression with a Lava C.O.S. scanner, followed by milling of Lava Ultimate block (composite resin) in a milling center; CeRe-digital impression with a Cerec 3D Bluecam scanner, followed by milling of Lava Ultimate block in Cerec; CeDis-digital impression with a Cerec 3D Bluecam scanner, followed by milling of IPS e.max CAD block (lithium disilicate) in Cerec; and PresDis-impression with polyvinyl siloxane, inlay made using the lost wax technique and IPS e.max Press pressed ceramic (lithium disilicate). Marginal and internal adaptations were measured using the replica technique. The inlay adjustments were performed using diamond burs in a contra-angle hand piece, and the time for adjustment was recorded using a timer, in seconds. The tightness of the proximal contact was measured using standardized metal blades. The statistical analyses for marginal fit data showed that at the cervical edge, CeDis (177.8 µm) had greater misfit than CeRe (116.7 µm), while all the groups had similar adaptation at the occlusal edge. The groups had similar internal fit at the pulpal wall, while LaRe (104.7 µm) > CeDis (66.7 µm) = CeRe (76.7 µm) at the axial wall. The groups restored with lithium disilicate ceramic took more time for adjustment when compared to the resin restorative material. The lowest proximal contact, in micrometers, was seen in the CeRe group (8.8 µm).

Early biofilm colonization on polished- and glazed-zirconium ceramic surface. Preliminary results.

AIM: The purpose of this study was to evaluate the effect of ceramic surface polishing procedure on the early dental biofilm formation on zirconium ceramics. METHODS: Twenty samples (discs shape) of tetragonal zirconium polycrystal stabilized with yttrium ceramics (Y-TZP) for LAVA system were fabricated (5 mm diameter and 1.5 mm thickness). Two patients with high level of dental hygiene were selected for this study. Oral devices covering the crowns of the upper premolars and molars were fabricated for each participant. Glazed and polished samples of Y-TZP ceramics were fixed on the vestibular and palatal zones of the devices. After 20 min (8 samples) and 1 h (8 samples) in the oral environment, the samples were removed and analyzed in a scanning electron microscope. The surface topographies of 4 ceramic samples (2 glazed and 2 polished) were analyzed (control group: without exposition in oral environment). RESULTS: The glazed samples showed a more irregular surface than polished samples. Deposition of granular aggregates was verified on all the samples in the two times of the study analyzed. This granular material coated more intensely on irregular areas, and its thickness increased after 1 h. No difference was observed as to bacterial morphology in any time of the study. Cocci and rods-shaped prevailed. CONCLUSIONS: Glazed surfaces presented larger tendency to dental biofilm accumulation.

The FHIT gene at 3p14.2 is abnormal in breast carcinomas.

Chromosome 3p deletions in breast cancer have been detected at 3p12-p21 by cytogenetic and loss of heterozygosity studies. Recently, we have cloned the FHIT (fragile histidine triad) gene, located at 3p14.2. Abnormalities of the FHIT locus were found in many established cancer cell lines, and the gene was abnormally transcribed in primary tumors of the digestive tract and lung. In this report, we describe the analysis of breast cancer, cell lines, and primary tumors for alterations in transcription of the FHIT gene; about 20% of the samples exhibited altered transcripts. In most of the cases, aberrant transcripts were missing exons. Lack of expression of FHIT mRNA was observed in another 10% of primary tumor samples. These results suggest that alterations in the FHIT gene may play an important role in breast cancer tumorigenesis and suggest that the MIT gene product functions in the control of the tumorigenic phenotype in a large variety of human neoplasms.

Increased in vivo phosphorylation of ret tyrosine 1062 is a potential pathogenetic mechanism of multiple endocrine neoplasia type 2B.

Mutations of the Ret receptor tyrosine kinase are responsible for inheritance of multiple endocrine neoplasia (MEN2A and MEN2B) and familial medullary thyroid carcinoma syndromes. Although several familial medullary thyroid carcinoma and most MEN2A mutations involve substitutions of extracellular cysteine residues, in most MEN2B cases there is a methionine-to-threonine substitution at position 918 (M918T) of the Ret kinase domain. The mechanism by which the MEN2B mutation converts Ret into a potent oncogene is poorly understood. Both MEN2A and MEN2B oncoproteins exert constitutive activation of the kinase. However, the highly aggressive MEN2B phenotype is not supported by higher levels of Ret-MEN2B kinase activity compared with Ret-MEN2A. It has been proposed that Ret-MEN2B is more than just an activated Ret kinase and that the M918T mutation, by targeting the kinase domain of Ret, might alter Ret substrate specificity, thus affecting Ret autophosphorylation sites and the ability of Ret to phosphorylate intracellular substrates. We show that the Ret-MEN2B mutation causes specific potentiated phosphorylation of tyrosine 1062 (Y1062) compared with Ret-MEN2A. Phosphorylated Y1062 is part of a Ret multiple effector docking site that mediates recruitment of the Shc adapter and of phosphatidylinositol-3 kinase (PI3K). Accordingly, we show that Ret-MEN2B is more active than Ret-MEN2A in associating with She and in causing constitutive activation of the Ras/mitogen-activated protein kinase and PI3K/Akt cascades. We conclude that the MEN2B mutation specifically potentiates the ability of Ret to autophosphorylate Y1062 and consequently to couple to the Ras/mitogen-activated protein kinase and the PI3K/Akt pathways. The more efficient triggering of these pathways may account for the difference between MEN2A and MEN2B syndromes.

Production of transgenic mice expressing the Ki-ras oncogene under the control of a thyroglobulin promoter.

Transgenic mice have been generated bearing three fusion genes consisting of: (a) a 900-base pair rat thyroglobulin promoter followed by a gene coding for a chloramphenicol acetyl transferase activity; (b) the same promoter followed by the complementary DNA of the human activated Ki-ras oncogene; (c) a 2000-base pair rat thyroglobulin promoter followed by the complementary DNA of the human activated Ki-ras. We have shown that the 900-base pair rat thyroglobulin promoter is able to direct the expression of the reporter gene specifically in the thyroid gland of transgenic mice. The mice bearing the two Ki-ras constructs, which express the transgene in thyroid glands, show thyroid abnormalities, although at very low incidence. These lesions appear after a long latency and with a benign aspect, thus suggest that, in agreement with literature data on naturally occurring human thyroid tumors, the action of an activated ras gene is not sufficient to attain a complete malignant conversion of thyroid glands in vivo. However, ras expression in thyroid follicular cells represents a favorable ground for tumor development, as shown by the fact that goitrogen stimulation experiments increase the occurrence of tumors.

Thymosin beta-10 gene overexpression correlated with the highly malignant neoplastic phenotype of transformed thyroid cells in vivo and in vitro.

A subtractive thyroid cDNA library was constructed from two human thyroid carcinoma cell lines originating from an anaplastic carcinoma and a papillary thyroid carcinoma. The library was used to identify genes correlated with the progression to a highly malignant phenotype. The thymosin beta-10 gene was isolated and found to be expressed at much higher levels in the anaplastic cell line than in the papillary cells. The thymosin beta-10 gene was overexpressed in five carcinoma cell lines compared with normal thyroid tissue and normal thyroid primary culture cells. The highest expression occurred in the most malignant cell lines. Thymosin beta-10 gene expression was also increased in surgically removed human thyroid carcinomas and was highest in the anaplastic carcinomas. Thymosin beta-10 gene expression was correlated with the degree of the malignant phenotype also in rat thyroid cells transfected with cellular and viral oncogenes of different tumorigenicity. These results show that thymosin beta-10 overexpression is a general event of thyroid cell neoplastic transformation and suggest that the gene is involved in the progression of thyroid carcinogenesis. Finally, the thymosin beta-10 gene was located on chromosome 2q37 by fluorescence in situ hybridization analysis.

The ATM gene and susceptibility to breast cancer: analysis of 38 breast tumors reveals no evidence for mutation.

Heterozygosity for ataxia-telangiectasia (A-T), a cancer-prone recessive syndrome, has been associated with an increased risk of breast cancer. The gene for A-T (ATM) is located at chromosomal region 11q22-q23, a region of frequent loss of constitutional heterozygosity in breast and other tumors. Loss of constitutional heterozygosity at 1lq22-q23 was found in 47% of informative cases in the series of primary tumors analyzed in this study. To investigate the role of ATM in breast cancer, we have determined the complete genomic organization of the gene, developed an exon-scanning PCR single-strand conformation polymorphism (PCR-SSCP) assay for mutation detection of ATM, and screened 38 consecutive breast tumors for mutations using both genomic DNA- and cDNA-based assays. In addition to common ATM polymorphisms detected both in the coding sequence and in flanking introns, seven unique SSCP alleles were identified in six tumor DNAs. Sequence analysis of these alleles revealed rive nucleotide substitutions that were predicted to change the encoded amino acid. However, PCR-SSCP and nucleotide sequencing analysis of the paired blood samples and of an extended sample size of a total of 224 chromosomes indicated that these SSCP patterns represent constitutional rare polymorphisms with a frequency between 0.005 and 0.023. Because the majority of A-T mutations are null mutations and none of the ATM alleles found in breast cancer samples would lead to the truncation of the translation product, we conclude that, in this initial sample of sporadic breast cancer patients, there was no evidence for an increased number of A-T carriers. In addition, because no somatic mutations were found, our study rules out the ATM gene as the frequently altered tumor suppressor gene at 11q23.

Activated RET/PTC oncogene elicits immediate early and delayed response genes in PC12 cells.

The expression of the receptor-like tyrosine kinase RET is associated with tumors, tissues or cell lines of neural crest origin. In addition RET products (Ret) are involved in determining cell fate during the differentiation of the enteric nervous system and during renal organogenesis. However, as yet, no direct evidence exists to indicate that the Ret kinase activity might interfere in a specific way with cellular differentiation, or proliferation, of a neural crest derived cell line. By using two constitutively activated forms of RET (RET/PTC1 and RET/PTC3) in transient transfection experiments, we have obtained evidence that active RET could reprogramme the gene expression pattern in the rat pheochromocytoma PC12 cell line. Transcription driven by gene promoters, such as NGFI-A and vgf, which belong, respectively, to primary and delayed response genes to nerve growth factor (NGF), and by the neuron-specific enolase (NSE) promoter, is rapidly induced by the expression of activated RET oncogenes. This induction is not elicited in other non neural derived cell types tested. We also demonstrate that endogenous ras activity is required for RET induction of these neural markers. Finally, in the RET/PTC transfected PC12 cells, NGF is unable to induce further their transcription. This suggests that RET/PTC could share an intracellular signalling pathway with the NGF-receptor.

The RFG oligomerization domain mediates kinase activation and re-localization of the RET/PTC3 oncoprotein to the plasma membrane.

The RET/PTC3 oncogene arises from the fusion between the N-terminal encoding domain of the RFG gene and the tyrosine kinase encoding domain of RET receptor. RET/PTC3 is very frequent in papillary thyroid carcinomas, especially in children exposed to the Chernobyl accident. We have studied the functional consequences of the RFG-RET fusion. Here we show that the N-terminal coiled-coil domain of RGF mediates oligomerization and activation of the kinase and of the transforming capability of RET/PTC3. In addition, the RFG coiled-coil domain mediates a physical association between RET/PTC3 and RGF proteins, rendering RFG a bona fide substrate of RET/PTC3 kinase. Finally, we show that the coiled-coil domain of RGF is essential for the distribution of the RET/PTC3 protein at the membrane/particulate cell compartment level, where also most of the RFG protein is localized. We propose that fusion to the RFG coiled-coil domain provides RET kinase with a scaffold that mediates oligomerization and re-localization of the RET/PTC3 protein, a process that may be crucial for the signalling of this specific RET/PTC variant.

RET/PTC1 oncogene signaling in PC Cl 3 thyroid cells requires the small GTP-binding protein Rho.

Thyroid papillary carcinomas are characterized by RET/PTC rearrangements that cause the tyrosine kinase domain of the RET receptor to fuse with N-terminal sequences encoded by heterologous genes. This results in the aberrant expression of a ligand-independent and constitutively active RET kinase. We analysed actin reorganization induced by the RET/PTC1 oncogene in PC Cl 3 rat thyroid epithelial cells. Differently from oncogenes Src, Ras and Raf, RET/PTC1 caused actin filaments to form prominent stress fibers. Moreover, stress fibers were identified in human thyroid papillary carcinoma cell lines harboring RET/PTC1 rearrangements but not in thyroid carcinoma cells negative for RET/PTC rearrangements. RET/MEN 2A, a constitutively active but unrearranged membrane-bound RET oncoprotein, did not induce stress fibers in PC Cl 3 cells. Induction of stress fibers by RET/PTC1 was restricted to thyroid cells; it did not occur in NIH3T3 fibroblasts or MCF7 mammary cells. RET/PTC1-mediated stress fiber formation depended on Rho but not Rac small GTPase activity. In addition, inhibition of Rho, but not of Rac, caused apoptosis of RET/PTC1-expressing thyroid cells. We conclude that Rho is implicated in the actin reorganization and cell survival mediated by the chimeric RET/PTC1 oncogene in thyroid epithelial cells, both phenotypes being cell type- and oncogene type-specific.