Anticalins directed against the fibronectin extra domain B as diagnostic tracers for glioblastomas.

The standard of care for diagnosis and therapy monitoring of gliomas is magnetic resonance imaging (MRI), which however, provides only an indirect and incomplete representation of the tumor mass, offers limited information for patient stratification according to WHO-grades and may insufficiently indicate tumor relapse after antiangiogenic therapy. Anticalins are alternative binding proteins obtained via combinatorial protein design from the human lipocalin scaffold that offer novel diagnostic reagents for histology and imaging applications. Here, the Anticalins N7A, N7E and N9B, which possess exquisite specificity and affinity for oncofetal fibronectin carrying the extra domain B (ED-B), a well-known proangiogenic extracellular matrix protein, were applied for immunohistochemical studies. When investigating ED-B expression in biopsies from 41 patients with confirmed gliomas of WHO grades I to IV, or in non-neoplastic brain samples, we found that Anticalins specifically detect ED-B in primary glioblastoma multiforme (GBM; WHO IV) but not in tumors of lower histopathological grade or in tumor-free brain. In primary GBM samples, ED-B specific Anticalins locate to fibronectin-rich perivascular areas that are associated with angiogenesis. Anticalins specifically detect ED-B both in fixed tumor specimen and on vital cells, as evidenced by cytofluorometry. Beyond that, we labeled an Anticalin with the γ-emitter (123) I and demonstrated specific binding to GBM-tissue samples using in vitro autoradiography. Overall, our data indicate that ED-B specific Anticalins are useful tools for the diagnosis of primary GBM and related angiogenic sites, presenting them as promising tracers for molecular tumor imaging.

Activation of DNA damage response by antitumor therapy counteracts the activity of vinca alkaloids.

BACKGROUND/AIM: Anthracyclines have been proven able to reduce the activity of vinca alkaloids by induction of cell-cycle arrest. The present study aims at identifying the critical initiation steps of signal transduction which transduce the inhibitory effects on the cytotoxicity of vinca alkaloids. MATERIALS AND METHODS: Several new cytostatic drug classes were evaluated together with vincristine in tumor cell lines and patients' tumor cells. RNA interference was used for molecular analyses. RESULTS: Inhibition of vincristine was observed by all cytostatic drugs, which induced cell-cycle arrest. Knockdown of proteins of the DNA damage response ascribed the inhibitory effect to a common pathway involving Chk-1, p53 and p21. Upstream of Chk-1 signal transduction depended on both ATM and ATR for all drugs except methotrexate. CONCLUSION: We have identified critical signaling steps of the DNA damage response system activated by cytostatic drugs, which reduce the anti-tumor activity of vinca alkaloids. The obtained results encourage the development of novel therapeutic strategies to prevent pathway interactions based on the molecular understanding of drug action and drug-drug interactions.

Dantrolene rescues arrhythmogenic RYR2 defect in a patient-specific stem cell model of catecholaminergic polymorphic ventricular tachycardia.

Coordinated release of calcium (Ca(2+) ) from the sarcoplasmic reticulum (SR) through cardiac ryanodine receptor (RYR2) channels is essential for cardiomyocyte function. In catecholaminergic polymorphic ventricular tachycardia (CPVT), an inherited disease characterized by stress-induced ventricular arrhythmias in young patients with structurally normal hearts, autosomal dominant mutations in RYR2 or recessive mutations in calsequestrin lead to aberrant diastolic Ca(2+) release from the SR causing arrhythmogenic delayed after depolarizations (DADs). Here, we report the generation of induced pluripotent stem cells (iPSCs) from a CPVT patient carrying a novel RYR2 S406L mutation. In patient iPSC-derived cardiomyocytes, catecholaminergic stress led to elevated diastolic Ca(2+) concentrations, a reduced SR Ca(2+) content and an increased susceptibility to DADs and arrhythmia as compared to control myocytes. This was due to increased frequency and duration of elementary Ca(2+) release events (Ca(2+) sparks). Dantrolene, a drug effective on malignant hyperthermia, restored normal Ca(2+) spark properties and rescued the arrhythmogenic phenotype. This suggests defective inter-domain interactions within the RYR2 channel as the pathomechanism of the S406L mutation. Our work provides a new in vitro model to study the pathogenesis of human cardiac arrhythmias and develop novel therapies for CPVT.