The localization capability of human mesenchymal stem cell (hMSCs) to tumors offers an attractive possibility as a cellular vehicle delivery route of immunotherapeutic genes. The RheoSwitch Therapeutic System® (RTS®) platform allows for regulated expression following exposure and withdrawal of activator ligand (AL) veledimex (also known as INXN-1001). In this study, we first evaluated RTS® regulated IL-12 expression from adenovirally transduced hMSC to generate a genetically modified hMSC (GM-hMSC) that could be used as a vehicle for gene delivery with the added benefit of migration potential to the tumor for therapy. Following establishment of the cultures in vitro, hMSCs were phentoypically similar following adenoviral vector transduction to mock transduced hMSCs based upon marker expression, characterized by flow cytometry. Levels of human and mouse IL-12 secreted from the hMSCs transduced with Ad-RTS-hIL-12 and Ad-RTS-mIL-12 respectively, directly correlated with the viral vector doses (1K-20K vp/cell). hMSC transduction efficiency was ∼90% with an MOI of 20K/cell. In addition, sustained cell levels of IL-12 expression were observed up to 53 days following transduction when maintained in the presence of veledimex. Furthermore, cycling of in vitro exposure periods between veledimex and excipient demonstrated the ability for on/off/on and off/on/off kinetics of IL-12 expression by transduced hMSCs. We then expanded on the concept of RTS® regulated gene expression in GM-hMSCs using multigenic plasmid constructs that simultaneously expressed three immunomodulators - human IL-12, human IFNα, and a CTLA4 decoy - in single, dual or triple combinations. Transient transfection of the multieffector plasmid using the AMAXA nucleofector system resulted in RTS® regulated concomitant expression of all three effectors. hIL-12 and hIFNα were found to be fully bio-functional in their respective cell based functional assays; - hIL-12 increased IFNγ secretion from NK92 cells, hIFNα enhanced STAT1 reporter activity, and the CTLA4 decoy functional assay is in progress. Taken together, these in vitro studies highlight the potential use of MSCs for tumor-targeted delivery of single or multiple RTS® regulated cancer immunotherapies. Altogether, use of these novel regulated immunotherapeutic approaches could potentially be translated into an effective clinical regimen for a variety of cancers. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C234. Citation Format: Tim Chan, Anjali Prabhu, Anissa Elayadi, Lindsay Williams, Vernon Dailey, Kristi Elliot, Tracey Snipas, Jonathan Carson, Jonathan Lewis, Stephen Schauer, Daniel Bednarik, Jayson M. Rieger, Laurent M. Humeau, Thomas R. Reed. Regulated immunomodulators expression using the RheoSwitch Therapeutic System® platform in human mesenchymal stem cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C234.

In Saccharomyces cerevisiae, the base excision DNA repair (BER) pathway has been thought to involve only a multinucleotide (long-patch) mechanism (LP-BER), in contrast to most known cases that include a major single-nucleotide pathway (SN-BER). The key step in mammalian SN-BER, removal of the 5′-terminal abasic residue generated by AP endonuclease incision, is effected by DNA polymerase β (Polβ). Computational analysis indicates that yeast Trf4 protein, with roles in sister chromatin cohesion and RNA quality control, is a new member of the X family of DNA polymerases that includes Polβ. Previous studies of yeast trf4Δ mutants revealed hypersensitivity to methylmethane sulfonate (MMS) but not UV light, a characteristic of BER mutants in other organisms. We found that, like mammalian Polβ, Trf4 is able to form a Schiff base intermediate with a 5′-deoxyribose-5-phosphate substrate and to excise the abasic residue through a dRP lyase activity. Also like Polβ, Trf4 forms stable cross-links in vitro to 5′-incised 2-deoxyribonolactone residues in DNA. We determined the sensitivity to MMS of strains with a trf4Δ mutation in a rad27Δ background, in an AP lyase-deficient background (ogg1 ntg1 ntg2), or in a pol4Δ background. Only a RAD27 genetic interaction was detected: there was higher sensitivity for strains mutated in both TRF4 and RAD27 than either single mutant, and overexpression of Trf4 in a rad27Δ background partially suppressed MMS sensitivity. The data strongly suggest a role for Trf4 in a pathway parallel to the Rad27-dependent LP-BER in yeast. Finally, we demonstrate that Trf5 significantly affects MMS sensitivity and thus probably BER efficiency in cells expressing either wild-type Trf4 or a C-terminus-deleted form.

(51) International Patent Classification: (81) Designated States (unless otherwise indicated, for every C12N 9/14 (2006.01) kind of national protection available): AE, AG, AL, AM, AO, AT,AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ, CA, (21) International Application Number: CH, CN, CO, CR, CU, CZ, DE, DK, DM, DO, DZ, EC, EE, PCT/US2008/058531 EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, HR, HU, ID, IL, IN, IS, JP, KE, KG, KM, KN, KP, KR, KZ, LA, LC, (22) International Filing Date: 27 March 2008 (27.03.2008) LK, LR, LS, LT, LU, LY, MA, MD, ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM, PG, PH, (25) Filing Language: English PL, PT, RO, RS, RU, SC, SD, SE, SG, SK, SL, SM, SV, SY, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, (26) Publication Language: English ZA, ZM, ZW

The stress hormone epinephrine is known to elicit multiple systemic effects that include changes in cardiovascular parameters and immune responses. However, information about its direct action on cancer cells is limited. Here we provide evidence that epinephrine reduces sensitivity of cancer cells to apoptosis through interaction with beta(2)-adrenergic receptors. The antiapoptotic mechanism of epinephrine primarily involves phosphorylation and inactivation of the proapoptotic protein BAD by cAMP-dependent protein kinase. Moreover, BAD phosphorylation was observed at epinephrine concentrations found after acute and chronic psychosocial stress. Antiapoptotic signaling by epinephrine could be one of the mechanisms by which stress promotes tumorigenesis and decreases the efficacy of anti-cancer therapies.

Background. Probes that allow site-specific protein labeling have become critical tools for visualizing biological processes. Methods. Here we used phage display to identify a novel peptide sequence with nanomolar affinity for near infrared (NIR) (benz) indolium fluorochromes. The developed peptide sequence ("IQ-tag'') allows detection of NIR dyes in a wide range of assays including ELISA, flow cytometry, high throughput screens, microscopy, and optical in vivo imaging. Significance. The described method is expected to have broad utility in numerous applications, namely site-specific protein imaging, target identification, cell tracking, and drug development.

The response of tumor cells to the unusual form of DNA damage caused by topoisomerase poisons such as camptothecin (CPT) is poorly understood, and knowledge regarding which drugs can be effectively combined with CPT is lacking. To better understand the response of tumor cells to CPT and to identify potential targets for adjuvant therapy, we examined global changes in mRNA abundance in HeLa cells after CPT treatment using Affymetrix U133A GeneChips, which include all annotated human genes (22,283 probe sets). Statistical analysis of the data using a Bayesian/ Cyber t test and a modified Benjamini and Hochberg correction for multiple hypotheses testing identified 188 probe sets that are induced and 495 that are repressed 8 h after CPT treatment at a False Discovery Rate of <0.05 and a minimum 3-fold change. This pharmacogenomic approach led us to identify two pathways that are CPT induced: (a) the epidermal growth factor receptor; and (b) nuclear factor-kappaB-regulated antiapoptotic factors. Experiments using HeLa cells in our lab and prior animal model studies performed elsewhere confirm that inhibitors of these respective pathways super-additively enhance CPT's cytotoxicity, suggesting their potential as targets for adjuvant therapy with CPT.

Release of cytochrome c from mitochondria to cytosol has been identified as one of the central events of apoptosis. Direct injection of cytochrome c induces apoptosis in some but not in all cell types. We observed that LNCaP prostate cancer cells failed to undergo apoptosis induced by cytochrome c microinjections. Microinjection of cytochrome c with another mitochondrial protein, Smac, was sufficient to activate caspases, however. Smac is believed to function as a neutralizer of caspase inhibitors, and mass spectrometry analysis identified XIAP as a predominant Smac binding protein in LNCaP cells. These findings are consistent with a requirement for a release of Smac from mitochondria to enable caspase activation in prostate cells. Indeed, translocation of Smac from mitochondria to cytosol was observed in LNCaP cells that undergo apoptosis and was inhibited by epidermal growth factor, which is a survival factor for these cells. These results further emphasize the central role of mitochondria in the regulation of apoptosis in prostate cancer cells.

Survival of cancer cells in response to therapy, immune response, or metastasis depends on interactions between pro- and antiapoptotic signals. Two major proapoptotic pathways have been described: (a) a death receptor pathway; and (b) a mitochondrial pathway. We reported previously that Akt and the epidermal growth factor (EGF) receptor send separate, redundant survival signals that act to inhibit the mitochondrial proapoptotic pathway in prostate cancer LNCaP cells. However, it was unclear at what level the pro- and antiapoptotic signals interact in these cells, and it was also unclear whether these signals would inhibit the death receptor pathway. We found that EGF can protect LNCaP cells from apoptosis induced by LY294002 but not from tumor necrosis factor a (TNF-alpha)-induced apoptosis. Furthermore, TNF-alpha induced apoptosis under conditions in which Akt was active. Treatment with TNF-alpha resulted in activation of caspase 8 and cleavage of BID, which in turn induced cytochrome c release and caspase 9-dependent activation of effector caspases. Thus, proapoptotic signals induced by both TNF-alpha and LY294002 converge on mitochondria and trigger cytochrome c release. Because EGF can inhibit cytochrome c release induced by LY294002 but not cytochrome c release induced by TNF-alpha, we suggest that the EGF survival mechanism operates on the mitochondrial pathway at a site upstream of cytochrome c release. The ability of TNF-alpha to bypass survival signals from activated EGF receptor and Akt in prostate cancer cells makes death receptor signaling a promising avenue for therapeutic intervention.

Constitutive activation of the phosphatidylinositol 3'-kinase (PI3 kinase)-Akt/protein kinase B (PKB) "survival signaling" pathway is a likely mechanism by which many cancers become refractory to cytotoxic therapy. In LNCaP prostate cancer cells, the PTEN phosphoinositide phosphatase is inactivated, leading to constitutive activation of Akt/PKB and resistance to apoptosis. However, apoptosis and inactivation of Akt/PKB can be induced in these cells by treatment with PI3 kinase inhibitors. Surprisingly, androgen, epidermal growth factor, or serum can protect these cells from apoptosis, even in the presence of PI3 kinase inhibitors and without activation of Akt/PKB, indicating the activity of a novel, Akt/PKB-independent survival pathway. This pathway blocks apoptosis at a level prior to caspase 3 activation and release of cytochrome c from mitochondria.