Synaptotagmins (Syts) are transmembrane proteins with two Ca2+-binding C2 domains in their cytosolic region. Syt I, the most widely studied isoform, has been proposed to function as a Ca2+ sensor in synaptic vesicle exocytosis. Several of the twelve known Syts are expressed primarily in brain, while a few are ubiquitous (Sudhof, T.C., and J. Rizo. 1996. Neuron. 17: 379–388; Butz, S., R. Fernandez-Chacon, F. Schmitz, R. Jahn, and T.C. Sudhof. 1999. J. Biol. Chem. 274:18290–18296). The ubiquitously expressed Syt VII binds syntaxin at free Ca2+ concentrations ([Ca2+]) below 10 μM, whereas other isoforms require 200–500 μM [Ca2+] or show no Ca2+-dependent syntaxin binding (Li, C., B. Ullrich, Z. Zhang, R.G.W. Anderson, N. Brose, and T.C. Sudhof. 1995. Nature. 375:594–599). We investigated the involvement of Syt VII in the exocytosis of lysosomes, which is triggered in several cell types at 1–5 μM [Ca2+] (Rodríguez, A., P. Webster, J. Ortego, and N.W. Andrews. 1997. J. Cell Biol. 137:93–104). Here, we show that Syt VII is localized on dense lysosomes in normal rat kidney (NRK) fibroblasts, and that GFP-tagged Syt VII is targeted to lysosomes after transfection. Recombinant fragments containing the C2A domain of Syt VII inhibit Ca2+-triggered secretion of β-hexosaminidase and surface translocation of Lgp120, whereas the C2A domain of the neuronal- specific isoform, Syt I, has no effect. Antibodies against the Syt VII C2A domain are also inhibitory in both assays, indicating that Syt VII plays a key role in the regulation of Ca2+-dependent lysosome exocytosis.

Ionizing radiation is a non-specific but highly effective way to kill malignant cells. However, tumor recurrence sustained by a minor fraction of surviving tumor cells is a commonplace phenomenon caused by the activation of both cancer cell intrinsic resistance mechanisms, and also extrinsic intermediaries of therapy resistance, represented by non-malignant cells and structural components of the tumor stroma. The improved accuracy offered by advanced radiotherapy (RT)-technology permits reduced volume of healthy tissue in the irradiated field, and has been triggering an increase in the prescription of high-dose oligofractionated regimens in the clinics. Given the remarkable clinical success of high-dose RT and the current therapeutic shift occurring in the field, in this review we revise the existing knowledge on the effects that different radiation regimens exert on the different compartments of the tumor microenvironment, and highlight the importance of anti-tumor immunity and other tumor cell extrinsic mechanisms influencing therapeutic responses to high-dose radiation.

BackgroundCancer-Associated Fibroblasts (CAFs) are significant components of solid malignancies and play central roles in cancer sustainability, invasion and metastasis. In this study we have investigated the invasive capacity and matrix remodelling properties of human lung CAFs after exposure to ablative doses of ionizing radiation (AIR), equivalent to single fractions delivered by stereotactic ablative radiotherapy (SART) for medically inoperable stage-I/II non-small-cell lung cancers.MethodsCAFs were isolated from lung tumour specimens from 16 donors. Initially, intrinsic radiosensitivity was evaluated by checking viability and extent of DNA-damage response (DDR) at different radiation doses. The migrative and invasive capacities of CAFs were thereafter determined after a sub-lethal single radiation dose of 18 Gy. To ascertain the mechanisms behind the altered invasive capacity of cells, expression of matrix metalloproteinases (MMPs) and their endogenous inhibitors (TIMPs) were measured in the conditioned media several days post-irradiation, along with expression of cell surface integrins and dynamics of focal contacts by vinculin-staining.ResultsExposing CAFs to 1 × 18 Gy resulted in a potent induction of multiple nuclear DDR foci (> 9/cell) with little resolution after 120 h, induced premature cellular senescence and inhibition of the proliferative, migrative and invasive capacity. AIR promoted MMP-3 and inhibited MMP-1 appearance to some extent, but did not affect expression of other major MMPs. Furthermore, surface expression of integrins α2, β1 and α5 was consistently enhanced, and a dramatic augmentation and redistribution of focal contacts was observed.ConclusionsOur data indicate that ablative doses of radiation exert advantageous inhibitory effects on the proliferative, migratory and invasive capacity of lung CAFs. The reduced motility of irradiated CAFs might be a consequence of stabilized focal contacts via integrins.

Sun'lnlaryThis study was undertaken to determine the fate of circulating NH2-terminal propeptide of type I procollagen (PlNP) in rats. P, adiolabded PINP showed a biphasic serum decay curve after intravenous injection. 79% of the material disappeared from the blood during the initial c~-phase (tl/2c~ = 0.6 min), while the remaining 21% was eliminated with a tl/213 of 3.3 rain. The major site of uptake was the liver, 78, 1, and 21% of its radioactivity being recovered in isolated liver endothdial cells (LEC), Kupffer calls, and parenchymal calls, respectively. In LEC, fluorescently labeled PINP accumulated in small (0.1/zm) peripheral and larger (>0.1 #m) perinuclear vesicles within 10 rain at 37~ after a binding pulse at 4~ These grew in size with increasing chasing time, reaching a maximum diameter of 1/zm or more after 30 rain, and taking the shape of rings that were stained only along their periphery. At chase intervals exceeding 30 min, the size of the vesicles decreased, and after 60 min the stain appeared in smaller, densely stained perinudearly located vesicles. Degradation of tzsI-PINP to free smaller fragments and 12sI-was significant after 30 min. Only formaldehyde-treated albumin, acetylated LDL, polyinosinic acid and NH2-terminal propeptide of type III procollagen (PIIINP) competed with PlNP for uptake. These findings indicate that clearance of PINP and PIIINP, which are normal waste products generated in large quantities, is a physiological function of the scavenger receptor in LEC.T ype I collagen is the most abundant collagen species in soft tissues and accounts for >90% of the organic matrix of mineralized bone. It is synthesized in the form of a larger protein, type I procollagen, which contains large additional domains at both ends (1). These parts, known as the NH2-and COOH-terminal propeptides of type I procollagen, are removed by two spedfic proteinases in the extracdlular space. Proper cleavage of these precursor-specific parts of the molecule is a prerequisite for the appropriate assembly of type I collagen molecules into collagen fibrils and fibers (2).Although collagen makes up about 30% of all animal protein, literature dealing with the catabolism of this protein and its propeptides is not extensive. Since the serum concentration of these substances is very low, powerful mechanisms evidently exist to eliminate these molecules. NH2-terminal propeptide of type I procollagen (PINP) 1 exists in two forms 1 Abbreviations used in this paper: AcLDL, acetyhted light density lipoprotein; Kay, relative elution position; KC, Kapffer cell; LDL, light density lipoprotein; LEC, liver endothelial cell; PC, parenchymal ceU; PICP, COOH-terminal propeptide of type I procollagen; PlNP, NH2-terminal propeptide of type I procollagen; PIIINP, NHz-terminal propeptide of type III procollagen; Poly I, polyinosinic acid; TC, tyraminyl cellbiose. in serum: the native trimeric PINP (35,000 Mr), and a smaller related to its NH2-terminal Coll domain (7,000 Mr), the former predominating. There is no solid evidence to...