Necrosis binding of Ac-Lys⁰(IRDye800CW)-Tyr³-octreotate: a consequence from cyanine-labeling of small molecules.

There is a growing body of nuclear contrast agents that are repurposed for fluorescence-guided surgery. New contrast agents are obtained by substituting the radioactive tag with, or adding a fluorescent cyanine to the molecular structure of antibodies or peptides. This enables intra-operative fluorescent detection of cancerous tissue, leading to more complete tumor resection. However, these fluorescent cyanines can have a remarkable influence on pharmacokinetics and tumor uptake, especially when labeled to smaller targeting vectors such as peptides. Here we demonstrate the effect of cyanine-mediated dead cell-binding of Ac-Lys <sup>0</sup> (IRDye800CW)-Tyr <sup>3</sup> -octreotate (800CW-TATE) and how this can be used as an advantage for fluorescence-guided surgery. Binding of 800CW-TATE could be blocked with DOTA <sup>0</sup> -Tyr <sup>3</sup> -octreotate (DOTA-TATE) on cultured SSTR <sub>2</sub> -positive U2OS cells and was absent in SSTR <sub>2</sub> negative U2OS cells. However, strong binding was observed to dead cells, which could not be blocked with DOTA-TATE and was also present in dead SSTR <sub>2</sub> negative cells. No SSTR <sub>2</sub> -mediated binding was observed in frozen tumor sections, possibly due to disruption of the cells in the process of sectioning the tissue before exposure to the contrast agent. DOTA-TATE blocking resulted in an incomplete reduction of 61.5 ± 5.8% fluorescence uptake by NCI-H69-tumors in mice. Near-infrared imaging and dead cell staining on paraffin sections from resected tumors revealed that fluorescence uptake persisted in necrotic regions upon blocking with DOTA-TATE. This study shows that labeling peptides with cyanines can result in dead cell binding. This does not hamper the ultimate purpose of fluorescence-guided surgery, as necrotic tissue appears in most solid tumors. Hence, the necrosis binding can increase the overall tumor uptake. Moreover, necrotic tissue should be removed as much as possible: it cannot be salvaged, causes inflammation, and is tumorigenic. However, when performing binding experiments to cells with disrupted membrane integrity, which is routinely done with nuclear probes, this dead cell-binding can resemble non-specific binding. This study will benefit the development of fluorescent contrast agents

Changes in the ornithine cycle following ionising radiation cause a cytotoxic conditioning of the culture medium of H35 hepatoma cells

Cultured H35 hepatoma cells release a cytotoxic factor in response to irradiation with X-rays. When the conditioned medium from irradiated cells is given to nonirradiated cells, growth is inhibited and followed by cell death, possibly apoptosis, Analysis of the conditioned medium reveals a dramatic change in the ornithine (urea) cycle components after the irradiation. A strong decrease in medium arginine is accompanied with parallel increases in ornithine, citrulline and ammonia. The high level of ammonia appears to be largely responsible for the observed cytotoxicity. The development of hyperammonia by irradiated cells and the related toxicity depend on the radiation dose and the number of cells seeded thereafter for the medium conditioning. Development of cytotoxicity by irradiated cells is completely prevented with the arginase inhibitor L-norvaline, in arginine-deficient medium or when citrulline replaces arginine. These preventive measures result in subtoxic ammonia levels

Toward a new generation of conditionally replicating adenoviruses:Pairing tumor selectivity with maximal oncolysis

Conditionally replicating adenoviruses (CRADs) represent a promising new platform for the treatment of cancer. CRADs have been demonstrated to kill tumor cells when other therapies fail, indicating that their antitumor properties are complementary to, and distinct from, those of standard treatments such as chemotherapy and radiation. In clinic trials CRADs have shown encouraging results, demonstrating mild side effects when administered at high doses and via different routes, including intratumorally, intraperitoneally, and intravenously. Tumor-selective replication has been detected, although as a single agent the efficacy appears to be limited. Interestingly, combined treatment with radiation or chemotherapy has been found to enhance CRAD efficacy considerably. To date, the molecular mechanisms underlying adenovirus-mediated oncolysis, and the way in which chemotherapy enhances oncolysis, are not well understood. A fuller knowledge of these processes will open up new strategies to improve the cell-killing potential of CRADs. Here, we discuss several possibilities that may lead to CRADs with enhanced oncolytic activity. These approaches include strategies to functionally couple tumor targeting and optimal oncolytic activity, and ways to further increase tumor cell disruption at later stages of infection to facilitate the spreading of virus throughout the tumor mass. In addition, improved methods to evaluate the efficacy of these agents in animal models, and in the clinic, will be required to systematically test and optimize CRAD efficacy, also taking into account the influence of tumor characteristics and the administration route

The Fanconi anemia proteins FAA and FAC function in different cellular compartments to protect against cross-linking agent cytotoxicity

Fanconi anemia (FA) is an autosomal recessive disease characterized by chromosomal instability, bone marrow failure, and a high risk of developing malignancies, Although the disorder is genetically heterogeneous, all FA cells are defined by their sensitivity to the apoptosis-inducing effect of cross-linking agents, such as mitomycin C (MMC), The cloned FA disease genes, FAC and FAA, encode proteins with no homology to each other or to any known protein. We generated a highly specific antibody against FAA and found the protein in both the cytoplasm and nucleus of mammalian cells. By subcellular fractionation, FAA is also associated with intracellular membranes, To identify the subcellular compartment that is relevant for FAA activity, we appended nuclear export and nuclear localization signals to the carboxy terminus of FAA and enriched its localization in either the cytoplasm or the nucleus. Nuclear localization of FAA was both necessary and sufficient to correct MMC sensitivity in FA-A cells. In addition, we found no evidence for an interaction between FAA and FAC either in vivo or in vitro. Together with a previous finding that FAC is active in the cytoplasm but not in the nucleus, our results indicate that FAA and FAC function in separate subcellular compartments. Thus, FAA and FAG, if functionally linked, are more likely to be in a linear pathway rather than form a macromolecular complex to protect against cross-linker cytotoxicity. (C) 1998 by The American Society of Hematology