Abstract LB-103: Circulating chromatin is a novel DNA damaging agent that induces genomic instability and malignant transformation
Authors: Mittra Indraneel , Mishra Pradyumna Kumar , Mansoor Shazia , Samant Urmila , Patkar Vivek , Sharma Suvarna , Ali Mansoor , Padhy Lakshmi Charan 
Published: February 2011

Abstract
A paradox of clinical oncology is that while chemotherapy (CT) or radiotherapy (RT) can induce dramatic tumor responses, they do not translate into commensurate improvements in patient survival. To address this paradox, we have proposed a hypothesis that posits that apoptotic genetic material released from tumor cells into the circulation can enter healthy cells at distant sites to induce oncogenic transformation that masquerade as metastasis (Nature Clinical Practice Oncology 2007;4:203). Implicit in this hypothesis is the suggestion that CT and RT, by inducing tumor cell apoptosis, may encourage dissemination of cancer by a process of “remote de novo oncogenesis”.
To test this hypothesis, we purified circulating chromatin fragments (PCFs) from sera of 40 cancer patients, both before and after CT or RT, and from 40 age and sex matched healthy donors, and added them to cells in culture. PCFs from all samples tested readily entered the recipients and triggered a DNA damage response (DDR) evidenced by the activation of H2AX and proteins of DDR and apoptotic pathways, namely, ataxia telangiectasia mutated (ATM), ataxia telangiectasia and Rad3 related (ATR), p53 and Caspase3. The DDR induced the integration of the incoming PCFs into the mouse cell genomes, and some of the genes contained within the PCFs were found to be expressed as human proteins. The extent of DDR induction by PCFs from the various samples followed the order: post- CT/RT &gt; pre- CT/RT &gt; healthy donors. When diverse cell types of human and mouse origin were exposed to PCFs, DDR was activated in all of them indicating that this activation was not cell-type specific but was universal in nature. When injected into mice, PCFs got stably integrated into the genomes of cells of their vital organs. Cultured cells exposed to PCFs showed numerous chromosomal aberrations as well as numerical increase and amplification of centrosomes within 48 hours, indicating an early onset of chromosomal instability. The treated cells exhibited a sequence of morphological changes within a span of 6 days that included: cell cycle arrest increase in cell size apoptosis senescence. By day 8, rapidly dividing resurgent cells with altered morphologies, some of which were apparently oncogenically transformed, arose to surround the senescent cells and ultimately to fill the dish. In contrast to PCFs, DNA purified from PCFs, or directly from serum, had no biological activity. When PCF-treated cells were injected into immuno-deficient mice, tumors developed in 26 % of the injected animals.
Our results clearly identify circulating chromatin fragments as novel DNA damaging agents that are capable of entering and integrating into healthy cell genomes to induce chromosomal instability and malignant transformation. These novel findings provide strong support for our proposal for a mechanism of metastasis that implicates a process of “remote de novo oncogenesis.”
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 LB-103.