Interference with DNA repair after ionizing radiation by a pyrrole-imidazole polyamide.

Pyrrole-imidazole (Py-Im) polyamides are synthetic non-genotoxic minor groove-binding small molecules. We hypothesized that Py-Im polyamides can modulate the cellular response to ionizing radiation. Pre-treatment of cells with a Py-Im polyamide prior to exposure to ionizing radiation resulted in a delay in resolution of phosphorylated γ-H2AX foci, increase in XRCC1 foci, and reduced cellular replication potential. RNA-sequencing of cell lines exposed to the polyamide showed induction of genes related to the ultraviolet radiation response. We observed that the polyamide is almost 10-fold more toxic to a cell line deficient in DNA ligase 3 as compared to the parental cell line. Alkaline single cell gel electrophoresis reveals that the polyamide induces genomic fragmentation in the ligase 3 deficient cell line but not the corresponding parental line. The polyamide interferes directly with DNA ligation in vitro. We conclude that Py-Im polyamides may be further explored as sensitizers to genotoxic therapies.

Wrong to be Right: Margin Laterality is an Independent Predictor of Biochemical Failure After Radical Prostatectomy.

OBJECTIVES: To examine the impact of positive surgical margin (PSM) laterality on failure after radical prostatectomy (RP). A PSM can influence local recurrence and outcomes after salvage radiation. Unlike intrinsic risk factors, a PSM is caused by intervention and thus iatrogenic failures may be elucidated by analyzing margin laterality as surgical approach is itself lateralized. PATIENTS AND METHODS: We reviewed 226 RP patients between 1991 and 2013 with PSM. Data includes operation type, pre/postoperative PSA, surgical pathology, and margin type (location, focality, laterality). The median follow-up was 47 months. Biochemical recurrence after RP was defined as PSA≥0.1 ng/mL or 2 consecutive rises above nadir. Ninety-two patients received salvage radiation therapy (SRT). Failure after SRT was defined as any PSA≥0.2 ng/mL or greater than presalvage. Kaplan-Meier and Cox multivariate analyses compared relapse rates. RESULTS: The majority of PSM were iatrogenic (58%). Laterality was associated with differences in median relapse: right 20 versus left 51 versus bilateral 14 months (P<0.01). Preoperative PSA, T-stage, Gleason grade, and laterality were associated with biochemical progression on univariate and multivariate analyses. Right-sided margins were more likely to progress than left (hazard ratio, 1.67; P=0.04). More right-sided margins were referred for SRT (55% right vs. 23% left vs. 22% bilateral), but were equally salvaged. Only T-stage and pre-SRT PSA independently influenced SRT success. CONCLUSIONS: Most PSM are iatrogenic, with right-sided more likely to progress (and sooner) than left sided. Margin laterality is a heretofore unrecognized independent predictor of biochemical relapse and hints at the need to modify the traditional unilateral surgical technique.

Size-selective collection of circulating tumor cells using Vortex technology.

A blood-based, low cost alternative to radiation intensive CT and PET imaging is critically needed for cancer prognosis and management of its treatment. "Liquid biopsies" of circulating tumor cells (CTCs) from a relatively non-invasive blood draw are particularly ideal, as they can be repeated regularly to provide up to date molecular information about the cancer, which would also open up key opportunities for personalized therapies. Beyond solely diagnostic applications, CTCs are also a subject of interest for drug development and cancer research. In this paper, we adapt a technology previously introduced, combining the use of micro-scale vortices and inertial focusing, specifically for the high-purity extraction of CTCs from blood samples. First, we systematically varied parameters including channel dimensions and flow rates to arrive at an optimal device for maximum trapping efficiency and purity. Second, we validated the final device for capture of cancer cell lines in blood, considering several factors, including the effect of blood dilution, red blood cell lysis and cell deformability, while demonstrating cell viability and independence on EpCAM expression. Finally, as a proof-of-concept, CTCs were successfully extracted and enumerated from the blood of patients with breast (N = 4, 25-51 CTCs per 7.5 mL) and lung cancer (N = 8, 23-317 CTCs per 7.5 mL). Importantly, samples were highly pure with limited leukocyte contamination (purity 57-94%). This Vortex approach offers significant advantages over existing technologies, especially in terms of processing time (20 min for 7.5 mL of whole blood), sample concentration (collecting cells in a small volume down to 300 µL), applicability to various cancer types, cell integrity and purity. We anticipate that its simplicity will aid widespread adoption by clinicians and biologists who desire to not only enumerate CTCs, but also uncover new CTC biology, such as unique gene mutations, vesicle secretion and roles in metastatic processes.

Arachidonate 5 lipoxygenase expression in papillary thyroid carcinoma promotes invasion via MMP-9 induction.

Arachidonate 5-lipoxygenase (ALOX5) expression and activity has been implicated in tumor pathogenesis, yet its role in papillary thyroid carcinoma (PTC) has not been characterized. ALOX5 protein and mRNA were upregulated in PTC compared to matched, normal thyroid tissue, and ALOX5 expression correlated with invasive tumor histopathology. Evidence suggests that PTC invasion is mediated through the induction of matrix metalloproteinases (MMPs) that can degrade and remodel the extracellular matrix (ECM). A correlation between MMP-9 and ALOX5 protein expression was established by immunohistochemical analysis of PTC and normal thyroid tissues using a tissue array. Transfection of ALOX5 into a PTC cell line (BCPAP) increased MMP-9 secretion and cell invasion across an ECM barrier. The ALOX5 product, 5(S)-hydroxyeicosatetraenoic acid also increased MMP-9 protein expression by BCPAP in a dose-dependent manner. Inhibitors of MMP-9 and ALOX5 reversed ALOX5-enhanced invasion. Here we describe a new role for ALOX5 as a mediator of invasion via MMP-9 induction; this ALOX5/MMP9 pathway represents a new avenue in the search for functional biomarkers and/or potential therapeutic targets for aggressive PTC.

Inhibition of uPAR and uPA reduces invasion in papillary thyroid carcinoma cells.

OBJECTIVES/HYPOTHESIS: We analyzed the expression of urokinase plasminogen activator (uPA) and its receptor (uPAR) in papillary thyroid carcinoma (PTC) and normal thyroid tissue and examined in vitro how uPA and uPAR contribute to an invasive/metastatic phenotype, and the functional consequences of inhibiting this system. STUDY DESIGN: Retrospective chart review of PTC patients, followed by prospective study using previously obtained patient tissue and PTC cellular models. METHODS: uPA and uPAR RNA and protein levels were analyzed in PTC patient tissue samples, PTC and normal thyroid tissue culture cells, and conditioned media (CM) using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and/or Western blotting. The plasminogen-activating ability of CM was examined using dark-quenched casein fluorimetry and casein-plasminogen gel zymography. The invasive potentials of the PTC and normal thyroid epithelial cell lines were assessed using an in vitro cellular invasion/migration system. RESULTS: uPA and uPAR RNA and protein levels were increased in PTC patient samples and PTC cells relative to controls. uPA and uPAR RNA were also significantly higher in patients with metastatic disease. Casein-plasminogen zymography and Western blotting demonstrated increased active uPA secreted by PTC cells compared with normal thyroid cells. Fluorimetric assays revealed that the PTC cells' CM was able to activate plasminogen, resulting in measurable casein hydrolysis. This casein hydrolysis was prevented by the addition of several specific uPA inhibitors. Finally, the in vitro invasion phenotypes of PTC cells were augmented by the addition of plasminogen, and this augmentation was reversed by inhibitory anti-uPA and anti-uPAR antibodies. CONCLUSIONS: These data provide new functional evidence of the uPA/uPAR system's role in PTC invasion/metastasis and demonstrate the attractiveness of uPA and uPAR as molecular biomarkers and therapeutic targets.

Dynamic changes in transcription factor complexes during erythroid differentiation revealed by quantitative proteomics.

During erythroid differentiation, beta-globin gene expression is regulated by the locus control region (LCR). The transcription factor NF-E2p18/MafK binds within this region and is essential for beta-globin expression in murine erythroleukemia (MEL) cells. Here we use the isotope-coded affinity tag (ICAT) technique of quantitative mass spectrometry to compare proteins interacting with NF-E2p18/MafK during differentiation. Our results define MafK as a 'dual-function' molecule that shifts from a repressive to an activating mode during erythroid differentiation. The exchange of MafK dimerization partner from Bach1 to NF-E2p45 is a key step in the switch from the repressed to the active state. This shift is associated with changes in the interaction of MafK with co-repressors and co-activators. Thus, our results suggest that in addition to its role as a cis-acting activator of beta-globin gene expression in differentiated erythroid cells, the LCR also promotes an active repression of beta-globin transcription in committed cells before terminal differentiation.