The CGL1 human hybrid cell system has been utilized for many decades as an excellent cellular tool for investigating neoplastic transformation. Substantial work has been done previously implicating genetic factors related to chromosome 11 to the alteration of tumorigenic phenotype in CGL1 cells. This includes candidate tumor suppressor gene FOSL1, a member of the AP-1 transcription factor complex which encodes for protein FRA1. Here we present novel evidence supporting the role of FOSL1 in the suppression of tumorigenicity in segregants of the CGL1 system. Gamma-induced mutant (GIM) and control (CON) cells were isolated from 7 Gy gamma-irradiated CGL1s. Western, Southern and Northern blot analysis were utilized to assess FOSL1/FRA1 expression as well as methylation studies. GIMs were transfected to re-express FRA1 and in vivo tumorigenicity studies were conducted. Global transcriptomic microarray and RT-qPCR analysis were used to further characterize these unique cell segregants. GIMs were found to be tumorigenic in vivo when injected into nude mice whereas CON cells were not. GIMs show loss of Fosl/FRA1 expression as confirmed by Western blot. Southern and Northern blot analysis further reveals that FRA1 reduction in tumorigenic CGL1 segregants is likely due to transcriptional suppression. Results suggest that radiation-induced neoplastic transformation of CGL1 is in part due to silencing of the FOSL1 tumor suppressor gene promoter by methylation. The radiation-induced tumorigenic GIMs transfected to re-express FRA1 resulted in suppression of subcutaneous tumor growth in nude mice in vivo. Global microarray analysis and RT-qPCR validation elucidated several hundred differentially expressed genes. Downstream analysis reveals a significant number of altered pathways and enriched Gene Ontology terms genes related to cellular adhesion, proliferation, and migration. Together these findings provide strong evidence that FRA1 is a tumor suppressor gene deleted and epigenetically silenced after ionizing radiation-induced neoplastic transformation in the CGL1 human hybrid cell system.

The financial crisis of 2008 is considered one of the most serious US economic downturns in the past 100 years, exceeded in severity only by the Great Depression of the 1930s. It included the collapses of a number of large financial institutions, government bailouts of many banks, a large drop in stock prices, and severe losses in the housing markets, associated with high rates of eviction and foreclosure. The wealth of Americans fell by trillions of dollars. People around the world are still laboring in its aftermath. The Levin-Coburn report of the US Congress found that the crisis resulted not from some unpredictable and unavoidable natural disaster but from complex and high-risk financial instruments [ 1 Coburn T. Levin C. Wall Street and the financial crisis: anatomy of a financial collapse. Permanent Subcommittee on Investigations, Washington, DC: US Senate2011 Google Scholar ]. This financial crisis illustrates 5 important professional lessons to which physicians and medical organizations need to attend if we are to avoid similar problems in our own ranks.

Mendonca, M. S., Chin-Sinex, H., Dhaemers, R., Mead, L. E., Yoder, M. C. and Ingram, D. A. Differential Mechanisms of X-Ray-Induced Cell Death in Human Endothelial Progenitor Cells Isolated from Cord Blood and Adults. Radial. Res. 176, 208-216 (2011).Endothelial colony-forming cells (ECFCs) are endothelial progenitor cells that circulate at low concentration in human umbilical cord and adult peripheral blood and are largely resident in blood vessels. ECFCs not only appear to be critical for normal vascular homeostasis and repair but may also contribute to tumor angiogenesis and response to therapy. To begin to characterize the potential role of ECFCs during the treatment of tumors in children and adults with radiation, we characterized the X-ray sensitivity of cord and adult blood-derived ECFCs. We found both cord blood and adult ECFCs to be highly radiation sensitive (3 Gy resulted in > 90% killing without induction of apoptosis). The X-ray survival curves suggested reduced potential for repair capacity, but X-ray fractionation studies demonstrated that all the ECFCs exhibited repair when the radiation was fractionated. Finally, the mechanisms of X-ray-induced cell death for cord blood and adult ECFCs were different at low and high dose. At low dose, all ECFCs appear to die by mitotic death/catastrophe. However, at high radiation doses (>= 10Gy) cord blood ECFCs underwent p53 stabilization and Bax-dependent apoptosis as well as p21-dependent G(1) and G(2)/M cell cycle checkpoints. By contrast, after 10 Gy adult ECFCs undergo only large-scale radiation-induced senescence, which is a cellular phenotype linked to premature development of atherosclerosis and vasculopathies. These data demonstrate that the ECFC response to radiation is dose-dependent and developmentally regulated and may provide potential mechanistic insight into their role in tumor and normal tissue response after ionizing radiation treatment. 2011 by Radiation Research Society

Abstract Treatment induced secondary malignancies after successful radiation and/or chemotherapy cure of primary tumors is a growing area of concern. The identification of chemopreventive compounds that reduce radiation-induced secondary malignancy but are both nontoxic and tolerated with long-term use is a critical need. We investigated the chemopreventive potential and mechanism of action of Vitamin E and EGCG (the active agent in green tea) with the human CGL1 radiation neoplastic transformation assay. The molecularly characterized CGL1 assay allows both alterations of the quantitative neoplastic transformation as well the potential underlying molecular processes of the chemoprevention to be determined. We found long-term treatment with 15 microM EGCG or 50 microM Vitamin E beginning 72 hours after 7 Gy irradiation, significantly reduce radiation-induced neoplastic transformation frequency by a factor of 2.3. We determined that Vit E suppressed radiation-induced carcinogenesis by the induction of a p53 and pro-apoptotic Bax dependent apoptosis in the progeny of the irradiated cells. In addition, we demonstrated that EGCG does not reduce radiation-induced carcinogenesis by increased apoptosis, but rather by the onset of p16 dependent senescence in the irradiated CGL1 progeny. Vit E and EGCG are excellent candidate nontoxic chemopreventive agents for radiation-induced carcinogenesis that work by two distinct molecular mechanisms. We propose that this information should also aid in the development of next generation chemopreventive compounds. These studies were supported by a grant from the DOD awarded to MSM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4368.

We investigated the efficacy and mechanism of dimethylaminoparthenolide (DMAPT), an NF-κB inhibitor, to sensitize human lung cancer cells to X-ray killing in vitro and in vivo. We tested whether DMAPT increased the effectiveness of single and fractionated X-ray treatment through inhibition of NF-κB and/or DNA double-strand break (DSB) repair. Treatment with DMAPT decreased plating efficiency, inhibited constitutive and radiation-induced NF-κB binding activity, and enhanced radiation-induced cell killing by dose modification factors of 1.8 and 1.4 in vitro. X-ray fractionation demonstrated that DMAPT inhibited split-dose recovery/repair, and neutral DNA comet assays confirmed that DMAPT altered the fast and slow components of X-ray-induced DNA DSB repair. Knockdown of the NF-κB family member p65 by siRNA increased radiation sensitivity and completely inhibited split-dose recovery in a manner very similar to DMAPT treatment. The data suggest a link between inhibition of NF-κB and inhibition of DSB repair by DMAPT that leads to enhancement of X-ray-induced cell killing in vitro in non-small-cell lung cancer cells. Studies of A549 tumor xenografts in nude mice demonstrated that DMAPT enhanced X-ray-induced tumor growth delay in vivo.

4619 The ability to isolate and characterize endothelial stem/progenitors should lead to a better understanding of their role in radiation-induced tumor response and normal tissue injury. We have characterized the X-ray sensitivity of four high proliferative potential endothelial stem/progenitor cell isolates from cord blood (CBM4, CBF10) and from adult peripheral blood (EPC060805 and LM2). Irradiation with 160 kVp X-rays revealed these cells to be all equally and highly radiation sensitive with 3 Gy killing > 90% of the cells, without the induction of apoptosis. However, at high radiation doses (10Gy or greater) despite extensive and equal cell killing, the cord blood endothelial progenitors underwent substantial apoptosis but the adult endothelial progenitors did not. In addition, after 10 Gy, a stabilization of DNA damage response p53 protein and an increase in the proapoptoic Bax protein were observed only in the cord blood endothelial progenitors. Furthermore, recent studies reveal that after 10 Gy instead of apoptosis the adult endothelial progenitors undergo radiation-induced senescence. Cell cycle and Western analysis revealed evidence of cell cycle checkpoints induction in both the cord blood and adult endothelial progenitors. The data indicate that while these endothelial progenitor cells are equally radiation sensitive their mode of cell death is dose and age dependent, and appears independent of cell cycle checkpoint function.