The serum ferritin concentration is a clinical parameter measured widely for the differential diagnosis of anemia. Its levels increase with elevations of tissue iron stores and with inflammation, but studies on cellular sources of serum ferritin as well as its subunit composition, degree of iron loading and glycosylation have given rise to conflicting results. To gain further understanding of serum ferritin, we have used traditional and modern methodologies to characterize mouse serum ferritin. We find that both splenic macrophages and proximal tubule cells of the kidney are possible cellular sources for serum ferritin and that serum ferritin is secreted by cells rather than being the product of a cytosolic leak from damaged cells. Mouse serum ferritin is composed mostly of L-subunits, whereas it contains few H-subunits and iron content is low. L-subunits of serum ferritin are frequently truncated at the C-terminus, giving rise to a characteristic 17-kD band that has been previously observed in lysosomal ferritin. Taken together with the fact that mouse serum ferritin is not detectably glycosylated, we propose that mouse serum ferritin is secreted through the nonclassical lysosomal secretory pathway. (Blood. 2010;116(9):1574-1584) IntroductionFerritin in mammals is a mostly intracellular, cytosolic iron storage and detoxification protein. It consists of H-and L-subunits that assemble into a 24-subunit hollow sphere in which iron is sequestered. 1 However, ferritin can also be detected extracellularly, in cerebrospinal fluid and sinovial fluid. In serum it is an extracellular ferritin that has been used extensively in diagnostic tests. In the clinical setting, serum ferritin evaluation is most commonly used to estimate body iron stores as low serum ferritin correlates with iron depletion, whereas high serum ferritin correlates with elevated body iron stores or with inflammation in patients with normal body iron stores. 2,3 Characterization of serum ferritin has produced many controversial results regarding subunit composition, iron content and other features. It has been compared in some studies to the "natural apoferritin" fraction found in many tissues, which is essentially devoid of iron 4 while other studies have claimed that serum ferritin contains considerable amounts of iron. 5 In insects, ferritin is a secreted protein composed of 2 types of subunits and the heteropolymer functions most likely as an iron transporter in the hemolymph. Each of the subunits has a classical secretion signal and ferritin is secreted from the insect fat body. 6 In contrast, mammalian H-and L-ferritin subunits lack signals mediating endoplasmic reticulum (ER) targeting, but ferritin can traffic to the lysosomal compartment through autophagy. 7 Binding of human serum ferritin to the lectin Concanavalin A (ConA) has previously suggested that ferritin is glycosylated 8,9 and actively secreted through the ER-Golgi pathway. 10 Indeed, it was shown in hepatocyte cell-models that inhibition of ER-Golgi trafficking abolished secretion o...

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Gene therapy clinical trials are limited due to several hurdles concerning the type of vector used, particularly, the viral vectors, and transfection efficacy when nonviral vectors are used. Therapeutic ultrasound is a promising non -viral technology that can be used in the clinical setting. Here, for the first time, we show the efficacy of therapeutic ultrasound to deliver genes encoding for hemopexin-like domain fragment (PEX), an inhibitor of angiogenesis, to prostate tumors in vivo. Moreover, the addition of an ultrasound contrast agent (Optison) to the transfection process was evaluated. Prostate cancer cells and endothelial cells (EC) were transfected in vitro with cDNA-PEX using therapeutic ultrasound alone (TUS + pPEX) or with Optison (TUS + pPEX + Optison). The biological activity of the expressed PEX was assessed using proliferation, migration, and apoptosis assays done on EC and prostate cancer cells. TUS + pPEX + Optison led to the inhibition of EC and prostate cancer cell proliferation (<65%), migration (<50%), and an increase in apoptosis. In vivo, C57/black mice were inoculated s.c. with prostate cancer cells. The tumors were treated with TUS + pPEX and TUS + pPEX + Optison either once or repeatedly. Tumor growth was evaluated, after which histology and immunohistochemistry analyses were done. A single treatment of TUS + pPEX led to a 35% inhibition in tumor growth. Using TUS + PEX + Optison led to an inhibition of 50%. Repeated treatments of TUS + pPEX + Optison were found to significantly (P < 0.001) inhibit prostate tumor growth by 80%, along with the angiogenic indices, with no toxicity to the surrounding tissues. These results depict the efficacy of therapeutic ultrasound as a non -viral technology to efficiently deliver genes to tumors in general, and to deliver angiogenic inhibitors to prostate cancer in particular. [Mol Cancer Ther 2007;6(8):2371 -82]