X-linked sideroblastic anemia with ataxia (XLSA/A) is caused by defects of the transporter ABCB7 and is characterized by mitochondrial iron deposition and excess of protoporphyrin in erythroid cells. We describe ABCB7 silencing in HeLa cells by performing sequential transfections with siRNAs. The phenotype of the ABCB7-deficient cells was characterized by a strong reduction in proliferation rate that was not rescued by iron supplementation, by evident signs of iron deficiency, and by a large approximately 6-fold increase of iron accumulation in the mitochondria that was poorly available to mitochondrial ferritin. The cells showed an increase of protoporphyrin IX, a higher sensitivity to H 2 O 2 toxicity, and a reduced activity of mitochondrial superoxide dismutase 2 (SOD2), while the activity of mitochondrial enzymes, such as citrate synthase or succinate dehydrogenase, and ATP content were not decreased. In contrast, aconitase activity, particularly that of the cytosolic, IRP1 form, was reduced. The results support the hypothesis that ABCB7 is involved in the transfer of iron from mitochondria to cytosol, and in the maturation of cytosolic Fe/S enzymes. In addition, the results indicate that anemia in XLSA/A is caused by the accumulation of iron in a form that is not readily usable for heme synthesis. IntroductionIron is essential in all eukaryotes for various vital functions including respiration, gene regulation, and DNA replication and repair. However, iron is also potentially toxic and dysregulation of its homeostasis may contribute to various hematologic, metabolic, and neurodegenerative diseases. 1 Mitochondria play a central role in iron metabolism, since the mitochondrion is the place of synthesis of heme and iron sulfur (Fe/S) proteins, 2 and dysregulation of mitochondrial iron is associated with certain diseases. 3 Among them is Friedreich ataxia, which is caused by deficiency of frataxin, a protein involved in mitochondrial iron trafficking 4 ; the X-linked sideroblastic anemia (XLSA) associated with deficiency of the erythroid-specific ALAS2 5 ; and XLSA/A, X-linked sideroblastic anemia with ataxia associated with defects of the ABC transporter ABCB7. 6 Most of our understanding of mitochondrial iron trafficking comes from the extensive studies done on S cerevisiae that showed that the organelle is the only site for the synthesis of Fe/S clusters, and that this activity is essential for the cell. 2,7 This biochemical pathway necessitates more than 10 different components, which include the cysteine desulphurase Nfs1p; the scaffold proteins Isu1p/Isu2p; chaperones; and the redox enzymes Arh1p, Yah1p, and glutaredoxin-5. 2 The functionality of Fe/S biosynthesis is essential also for the assembly of extramitochondrial Fe/S enzymes, including Leu1p and Rli1p, the latter involved in ribosome biogenesis 8,9 ; but biosynthesis requires a set of accessory proteins that include the ABC transporter named Atm1p, the sulphydryl oxidase Erv1p of mitochondrial intermembrane space, glutathione, the cytosolic P-lo...

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IntroductionWe investigated the frequency of detection and the prognostic and predictive significance of circulating tumor cells (CTCs) in patients with recurrent/metastatic (R/M) head and neck carcinoma (HNC) before starting systemic therapy.Patients and methodsUsing the CellSearch technology, CTCs were assessed prospectively in peripheral blood of 53 R/M-HNC patients. We performed spiking experiments to test the diagnostic performance of the CellSearch platform in identifying squamous carcinoma cells.ResultsCTCs were identified in 14 (26%) and 22 (41%) patients at baseline and at any time point, respectively. In univariate analysis ≥2 CTCs had a poorer prognostic role than 0–1 CTC. In multivariate analysis, the presence of one CTC or more was associated with a poor prognosis both in terms of progression-free survival (PFS) [Hazard Ratio (HR): 3.068, 95% confidence interval (CI): 1.53–6.13, p 0.002] and overall survival (OS) [HR: 3.0, 95% CI: 1.48–6.0, p 0.002]. A disease control after systemic therapy was obtained in 8% of CTC-positive patients as opposed to 45% in CTC-negative ones (p 0.03). The epidermal growth factor receptor (EGFR) expression was identified in 45% of CTC-positive patients.DiscussionIn conclusion, CTCs are detected in one out of three patients with RM-HNC. CTC detection is a strong prognostic parameter and may be predictive of treatment efficacy. The frequency of EGFR expression in CTCs seems to be lower than that expected in the primary tumor.

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