John Puskas

Photodynamic therapy mediates the oxygen-independent activation of hypoxia-inducible factor 1α

Photodynamic therapy (PDT) induces the expression of the hypoxia-inducible factor 1α (HIF-1α) subunit of the HIF-1 transcription factor and its target genes in vitro and in vivo. PDT also induces the expression of the enzyme cyclooxygenase-2 and its metabolite, prostaglandin E2 (PGE2). PGE2 and hypoxia act independently and synergistically to increase HIF-1α accumulation and nuclear translocation. To examine the expression of HIF-1 target genes in response to PDT-mediated oxidative stress and PGE2 under normoxic conditions, we established EMT6 cells transfected with a plasmid consisting of a hypoxia response element promoter and a downstream gene encoding for green fluorescent protein (GFP). To examine the temporal kinetics of HIF-1α nuclear translocation in response to PDT, we transfected a second line of EMT6 cells with a GFP-tagged HIF-1α fusion vector. Cell monolayers were incubated with 1 μg mL−1 Photofrin for 24 h and irradiated with fluences of 1, 2.5, and 5 J cm−2. Direct measurement of oxygen concentration during irradiation confirmed that cells remained well oxygenated. Cells were also exposed to 1 and 10 μmol/L PGE2 for 3 h. In normoxic conditions, Photofrin, PDT, and PGE2 treatment activated HIF-1α and induced its nuclear translocation. Maximal Photofrin-PDT–mediated HIF-1α activation was intermediate in magnitude between that induced by PGE2 and that by the hypoxia mimic cobalt chloride. This work establishes that PDT induces significant activation of the HIF-1α pathway in the absence of hypoxia and supports the interpretation that the induction of HIF-1 target genes by PDT may be mediated, at least in part, by the prostaglandin pathway. [Mol Cancer Ther 2006;5(12):3268–74]

Development of an attenuated interleukin-2 fusion protein that can be activated by tumour-expressed proteases

The ability to alter the cytokine microenvironment has the potential to shape immune responses in many physiological settings, including the immunotherapy of tumours. We set out to develop a general approach in which cytokines could be functionally attenuated until activated. We report the development and initial characterization of fusion proteins in which human or mouse interleukin‐2 (IL‐2), a potent growth factor for immune cells, is joined to a specific IL‐2 inhibitory binding component separated by a protease site. The rationale is that upon cleavage by a protease the cytokine is free to dissociate from the inhibitory component and becomes biologically more available. We describe the successful development of two attenuation strategies using specific binding: the first uses the mouse IL‐2 receptor alpha chain as the inhibitory binding component whereas the second employs a human antibody fragment (scFv) reactive with human IL‐2. We demonstrated that the fusion proteins containing a prostate‐specific antigen or a matrix metalloproteinase (MMP) protease cleavage site are markedly attenuated in the intact fusion protein but had enhanced bioactivity of IL‐2 in vitro when cleaved. Further, we showed that a fusion protein composed of the IL‐2/IL‐2 receptor alpha chain with an MMP cleavage site reduced tumour growth in vivo in a peritoneal mouse tumour model. This general strategy should be applicable to other proteases and immune modulators allowing site‐specific activation of immunomodulators while reducing unwanted side‐effects.

Detection of G Protein-Coupled Receptors by Immunofluorescence Microscopy

G protein-coupled receptors (GPCRs) are activated by a wide array of signals, which include light, neurotransmitters, hormones, cytokines, and drugs. Knowledge of the subcellular distribution of GPCRs is required in many experimental situations. Most GPCR signaling occurs in response to activators that interact with receptors localized on the cell surface. GPCRs must move from their site of synthesis to the plasma membrane, often undergoing redistribution in response to ligand binding. Endocytosis and recycling of receptors are important for desensitization and resensitization. Furthermore, mutations in GPCRs can alter receptor trafficking and prevent normal receptor function. This chapter describes a widely applicable method for visualizing a GPCR by indirect immunofluorescence microscopy.

Detection, molecular profiling and culture of CSF-CTCs in leptomeningeal disease (LMDz) in melanoma.

e21543Background: Approx. 5% of melanoma-associated brain tumor cases also develop LMDz. Patients with LMDz have devastating survival outcome, and currently there is no effective treatment. The aim...

Abstract 2108: Detection and molecular profiling of leptomeningeal disease in melanoma

Approximately 5% of melanoma patients develop leptomeningeal disease (LMDz), a highly symptomatic complication with a dismal survival of 8-10 weeks. The aim of this study was to determine whether diagnosis and personalized treatment for melanoma-LMDz could be improved by assessing patient-derived cerebrospinal fluid (CSF) specimens. Circulating tumor cells in CSF (CSF-CTCs) were detected by Veridex CellSearch® System and the circulating melanoma cell kit, based on anti-CD146 and anti-high molecular weight melanoma associated antigen (HMW-MAA-PE (MEL-PE)). Of the 12 patients with definitive LMDz diagnosis, all but 1 patient (92%) had CSF-CTCs (range 23-3055 CTCs/ml). In contrast, only 3/8 (37%) melanoma patients without LMDz diagnosis had CSF-CTCs detected, with significantly lower CTC counts per ml CSF (range 0.13-0.6 CTCs/ml). Ex vivo studies of CSF uncovered that although patient-derived CSF does not appear to act as a chemo-attractant or a stimulant of invasion to initiate melanoma migration to the leptomeninges, it significantly reduced the ability of BRAF inhibitors to induce apoptosis in established melanoma cell lines (p Citation Format: Inna Smalley, Brittany Evernden, Vincent Law, Rajappa Kenchappa, John Puskas, Elena Ryzhova, Nam Tran, Arnold Etame, Solmaz Sahebjam, Anthony Magliocco, Peter Forsyth, Keiran S. Smalley. Detection and molecular profiling of leptomeningeal disease in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2108.